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The H-NS protein, the product of the hns gene, plays a central role in the cellular response of bacteria to environmental stresses such as modification of osmolarity and temperature. The leucine regulatory protein (Lrp) controls a wide array of operons both as an activator (e.g. ilvIH) and as a repressor. We demonstrate that H-NS can decrease the activity of Lrp in stationary phase and under conditions of high osmolarity. Strains containing hns mutations have higher levels of Lrp-activated ilvIH transcription, while strains carrying the hns+ allele on a pBR322 plasmid have lower activity of Lrp-directed ilvIH gene expression.

The H-NS protein of Escherichia coli regulates the expression of genes involved in many general processes such as osmoregulation and virulence. More recently, H-NS was shown to exert an effect on ilvIH gene expression in conjunction with the leucine responsive regulatory protein (Lrp). We show that H-NS is involved in the transcriptional regulation of the kbl/tdh operon, which is also Lrp regulated. Insertional inactivation of the hns gene results in two-fold derepression of the kbl/tdh operon. This level of expression is sufficient to suppress the auxotrophic requirements imposed by a glyA mutation. We show that expression of the kbl/tdh operon is temperature controlled and that this control is not mediated through H-NS action as has been shown for some other temperature controlled genes.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.

We studied derivatives of Escherichia coli K-12 expressing only one of the three acetohydroxy acid synthases, acetohydroxy acid synthase I (the ilvB gene product), to gain insight into the mechanism of synthesis of this isoenzyme. We found that acetohydroxy acid synthase I is regulated by a multivalent control requiring leucine and valine. In fact, a starvation of each of these two amino acids causes a derepressed synthesis of acetohydroxy acid synthase I, while a starvation of isoleucine has no effect. Derepressed synthesis of this enzyme also occurs when cells are grown in the presence of α-ketobutyrate, unless the three branched-chain amino acids are also present. Additional studies with α-aminobutyric acid, an inhibitor of valyl-tRNA Val synthesis, show that valine must be charged to tRNA to participate in this regulatory system.

We isolated a strain (PS187) containing a mutation in the ilvA structural gene (threonine deaminase) affecting the regulation of the isoleucine and valine biosynthetic enzymes. We studied the effect of the mutation, ilvA538, on the ability of the ilvADGE operon to respond to limitations of branched chain amino acids. We found that this regulatory mutation prevents a derepression response to limitations of branched chain amino acids. The mutation is epistatic to constitutive mutations which are deficient in co-repressor synthesis. In addition, the ilvA+ allele is dominant to ilvA538 in trans, while a cis regulatory mutation, vlr-2005, is dominant in cis. We suggest a positive regulatory role for the ilvA gene product and present evidence that the apo-dimer form of threonine deaminase is the molecular species essential for an efficient depression response of the ilvADGE operon.

Some of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.

A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.

Acetohydroxy acid synthase III (AHAS III) is one of the three isoenzymes which catalyze the condensation reaction for the biosynthesis of the branched chain amino acids in Escherichia coli K-12. The synthesis of this enzyme is repressed by leucine. As a consequence of this regulatory feature, strain PS1035, in which AHAS III is the only AHAS isoenzyme expressed, does not grow in minimal medium containing leucine. The other two branched chain amino acids, isoleucine and valine, do not have regulatory effects on AHAS III synthesis.

We studied the properties of the two acetohydroxy acid synthase isoenzymes expressed in wild type Escherichia coli K-12 in two isogenic strains, PS1035 (containing only acetohydroxy acid synthase III) and PS1036 (containing only acetohydroxy acid synthase I). The pH dependence is different for the two enzymes: acetohydroxy acid synthase I shows optimum activity at neutral pH, while acetohydroxy acid synthase III is most active at alkaline pH. Both activities require Mg2+ and thiamine pyrophosphate, but acetohydroxy acid synthase I, as compared to acetohydroxy acid synthase III, has a specific requirement for flavin adenine dinucleotide. Acetohydroxy acid synthase I is also more resistant to valine inhibition but more sensitive to inactivating conditions such as dialysis and temperature. The catalytic role of acetohydroxy acid synthase I in the synthesis of α-acetolactate is characterized by a higher affinity for pyruvate and a lower sensitivity to inhibition by α-ketobutyrate.

We have studied the levels of the enzymes corresponding to the genes remaining in deletion mutations linking the histidine and rough B operons of Salmonella typhimurium. Such deletion mutations leave the two promoter regions intact, as the two operons are oriented in opposite directions. Derepression of the histidine operon leads to a fivefold decrease in the rate of synthesis of the enzyme corresponding to the first intact rfb gene adjacent to the end of the deletion. When the histidine operon is repressed, there is no inhibition of any rough B enzyme, but the rate of synthesis of the enzyme corresponding to the first intact his gene adjacent to the end of the deletion is decreased fourfold. Studies of the rate of synthesis of the his enzyme corresponding to the gene adjacent to the end of a deletion, at several levels of partial derepression, are consistent with a simple model of interference between the RNA transcription apparatus of the two operons.

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3'-phosphate-5'-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3',5'-diphosphate (PAP). Replacement of sulfate and ATP by this PAP(35)S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP(35)S to thiosulfate still requires the addition of Mg(2+) and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP(35)S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.

A cell-free system from Chlorella pyrenoidosa Chick (Emerson strain 3) which produces acid-volatile radioactivity from (35)SO(4) (2-) is described. A high speed supernatant from cells broken in the French Press at pH 7.0 shows maximal activity when fortified with ATP, an ATP-generating system (creatine phosphate and creatine phosphokinase), TPN, a TPN-reducing system (glucose-6-phosphate and glucose-6-phosphate dehydrogenase) and MgCl(2). This system is quite labile and is not stable to dialysis. Addition of low concentrations of 2,3,-dimercaptopropan-1-ol (BAL) to the buffers used for enzyme preparation stabilize the extracts and permit them to be dialyzed for 4 hours without loss of activity. If additional BAL is also added to the incubation mixtures it can replace TPNH as a reductant. DPNH also shows appreciable acticity.The system prepared with BAL-containing buffers shows maximal activity at pH 9.0. At this pH, the system requires only ATP, Mg(2+) and additional BAL and has high activity and stability compared with the other conditions tried. The optimum concentrations of these reactants has been determined and the kinetics of production of acid-volatile radioactivity are described. Nucleoside triphosphates other than ATP are not appreciably active in this system. In all cases, anaerobic conditions are required for maximal activity, the enzyme extracts are labile to heat, and no unequivocal requirement for thioctic acid can be demonstrated.

An ilvA mutation carried by a ø80i λ dilv transducing phage complemented some ilvA mutations and did not complement others. Complementation was accompanied by appearance of threonine deaminase activity in vivo. These results divided the ilvA mutations into two sets which formerly appeared to define two cistrons.

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 gworn 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.

The tdh operon of Escherichia coli consists of two genes whose products catalyze sequential steps in the formation of glycine and acetyl coenzyme A from threonine. The operation of the tdh pathway can potentially confer at least two capabilities on the cell: the first is to provide a biosynthetic source of glycine, serine, or both that is an alternative to the conventional (triose phosphate) pathway; the second is to enable cells to utilize threonine as the sole carbon source. The latter capability is referred to as the Tuc+ phenotype. In wild-type E. coli, the tdh operon is expressed at levels that are too low to bestow the Tuc+ phenotype, even in leucine-supplemented media, where the operon is induced eightfold. In eight Tuc+ mutants, the expression of the tdh operon was elevated 100-fold relative to the uninduced wild-type operon. The physical state of the DNA at the tdh locus in these Tuc+ strains was analyzed by Southern blotting and by DNA sequencing. In eight independent isolates the mobile genetic element IS3 was found to lie within the tdh promoter region in identical orientations. In six cases that were examined by DNA sequencing, IS3 occupied identical sites between the -10 and -35 elements of the tdh promoter. The transcription start points for the wild-type tdh promoter and one IS3-activated tdh promoter were identical. In effect, the repeatedly observed transposition event juxtaposed an IS3-borne -35 region and the tdh-specific -10 region, generating a hybrid promoter whose utilization led to elevated, constitutive expression of the tdh operon. This is the first case of promoter activation by IS3 where the site of transcription initiation is unaltered.

We describe the regulatory properties of two strains carrying either the ilvA624 or the ilvA625 mutations, located in the structural gene for threonine deaminase. Crude extracts of both these strains possess a threonine deaminase activity migrating on polyacrylamide gels, differently from the wild type enzyme. Growth studies demonstrate that these mutations do not cause a limitation of isoleucine biosynthesis, suggesting normal catalytic activity of deaminase. A regulatory consequence of the ilvA624 allele is a derepression of the isoleucine-valine biosynthetic enzymes, which is recessive to an ilvA+ allele. The ilvA625 mutation causes a derepression which is dominant in an ilvA625/ILVA+ diploid. We interpret these data assuming that threonine deaminase, previously shown to be an autogenous regulator of the ilv genes, lacks a repressor function in the ilvA624 mutant, while in the ilvA625 mutant it is a better activator than wild type threonine deaminase. The data are discussed in terms of a model requiring that threonine deaminase, or a precursor of it, is in equilibrium between two forms, one being an activator of gene expression and the other being a repressor.

The flow of carbon to alpha-acetolactate is Escherichia coli K12 is shown to involve the endogenous pool of alpha-ketobutyrate (alpha-KB). In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for alpha-KB sufficiently high that alpha-acetolactate production is severely limited when alpha K-B is supplied exogenously. The ability of threonine deaminase to make alpha-KB is correlated with the synthesis of the AHAS isoenzymes. Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect alpha-KB production and the expression of the AHAS isoenzymes in a direct way. The ilv A538 mutation results in a feedback-hypersensitive threonine deaminase ans slow alpha-KB and AHAS production. A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces alpha-KB and AHAS more quickly. A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase alpha-KB production in vivo. Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine. The physiological implications of the role of alpha-KB in the biosynthesis of acetolactate are discussed.

A spontaneous mutation of the pi -histidine plasmid has been isolated that causes increased expression of the histidine genes and the gnd gene which are normally present on the plasmid. Transduction frequency and F-mediated conjugational transfer of the mutant plasmid indicate gene amplification has occurred without an increase in the number of replicating units. A model for gene amplification dependent upon insertion sequences is proposed to explain the mutant phenotype.

Separation of the products formed from sulfate-(35)S by cell-free extracts of Chlorella pyrenoidosa (Emerson Strain 3) has permitted the identification of thiosulfate as a major product which yields acid-volatile radioactivity. The products formed, as separated by Dowex-1-nitrate chromatography, are qualitatively the same whether extracts at pH 7.0 (using TPNH as the reductant) or extracts at pH 9 [using 2,3-dimercaptopropan-1-ol, (BAL) as reductant] are employed. While thiosulfate can be separated without the addition of carrier, the inclusion of carrier improves the recovery. High concentrations of ATP which have been shown previously to inhibit the formation of acid-volatile radioactivity from radioactive sulfate, inhibit the formation of thiosulfate almost completely. Degradation of the thiosulfate formed at normal ATP concentrations reveals that most of the radioactivity is in the SO(3)-sulfur of the molecule suggesting that the SH-sulfur is derived from the enzyme extracts. If carrier sulfite is present during thiosulfate formation from sulfate-(35)S, radioactive sulfite is recovered at the expense of radioactive thiosulfate. Reconstruction experiments utilizing specifically-labeled thiosulfates indicate that radioactive sulfite formation is probably not the result of trapping a normal intermediate, but can be attributed to non-enzymatic exchange between labeled thiosulfate formed from sulfate and the non-radioactive sulfite added, suggesting that free sulfite is not an intermediate in thiosulfate formation from sulfate.

I identified two enzyme activities, α-galactosidase and a galactoside permease, required for melibiose metabolism by Salmonella typhimurium . These activities are very low under normal growth conditions, but their production can be induced by melibiose and gratuitously by melibiitol. Melibiose induction is severely inhibited by glucose, but the glucose effect can be countered by 3′, 5′ cyclic adenosine monophosphate. I isolated two phenotypic classes of mutants not able to utilize melibiose as a carbon source. One class, Car − , is deficient in the phosphotransferase system. The other, Mel, lacks either α-galactosidase, galactoside permease, or both functions.

The Salmonella typhimurium pi-histidine episome was identified by Ames et al. (2) in an unstable partial revertant of a deletion mutation-containing strain, hisG203. HisG203 lacks the histidine operator, promoter, and part of the first structural gene. In this paper, we study some properties of pi factor and demonstrate a low frequency of pi integration into the chromosome at or near the histidine region.

An ilv A gene (specifying threonine deaminase) mutation causes an Escherichia coli K12 strain to be leucine sensitive. The regulation of all the isoleucine and valine biosynthetic enzymes and of the branched chain aminoacyl-tRNA synthetases is affected in this mutant. We postulate that threonine deaminase is a positive effector in the multivalent control process.

Mutants ( car ) isolated from Salmonella typhimurium were unable to utilize or ferment the following carbohydrates (all d -configuration): glucose, fructose, mannose, N -acetylglucosamine, sorbitol, mannitol, maltose, melibiose, and glycerol. The mutants did utilize galactose, glucose 6-phosphate, gluconic acid, glucuronic acid, pyruvate, and l -lactate. Biochemical analysis showed that there were two classes of mutants, each lacking one component of a phosphotransferase system. CarA mutants were deficient in enzyme I; carB lacked the phosphate carrier protein, HPr. Mapping experiments showed that the carA gene was located near pro ; the carB gene mapped near purC .