Analysis of Polar and Nonpolar Tryptophan Mutants by Derepression Kinetics

A comparison of the rates of synthesis of the tryptophan biosynthetic enzymes of Salmonella typhimurium under derepression showed that the genes of the trp operon can be expressed in a coordinate fashion in auxotrophs carrying nonpolar mutations. This coordination disappeared in trpA polar mutants. The loss of coordination affected only trpB, the second gene in the operon, which was always more drastically affected than the three distal genes. Polar mutations in trpA, the first gene of the trp operon, reduced the rates of synthesis of the tryptophan biosynthetic enzymes under conditions of derepression. When these rates were measured and correlated with the map position of each polar mutation, a polarity gradient of decreasing intensity (moving distally from the operator end of the gene) was obtained. Certain mutations ("unusual mutations") mapping at the operator distal end of trpA, and considered by other workers to correspond to the operator proximal end of trpB, were found to be polar. The bearing of our observations on the question of coordinate versus semicoordinate expression of the trp genes and the status of the "unusual mutations" is discussed.     

Genetic Fine Structure of the Leucine Operon of Escherichia coli K-12

The order of mutational sites in 10 independently isolated leucine auxotrophys of Escherichia coli K-12 was determined by three-point reciprocal transductions. The sites of mutation mapped in linear sequence in a cluster; all leucine auxotrophic mutations were cotransducible with mutations in the arabinose operon. The mutations were assigned to four complementation groups by abortive transduction tests, designated D, C, B, and A, reading in a clockwise direction from the arabinose operon. Enzyme analyses showed that strains with a mutation in gene A lacked alpha-isopropylmalate synthetase activity (EC 4.1.3), and those with a mutation in gene B lacked beta-isopropylmalate dehydrogenase activity (EC 1.1.1). It is concluded that the gross structure of the leucine operon in E. coli is closely similar to, if not identical with, the gross structure of the leucine operon in Salmonella typhimurium.     

The Genetic Basis for Mucoidy and Radiation Sensitivity in capR (lon) Mutants of E. coli K-12

CapR mutants of E. coli K-12 overproduce capsular polysaccharide (mucoid phenotype) and enzymes involved in capsular polysaccharide synthesis, and they are sensitive to radiation. It has been uncertain whether both properties are mediated by damage to a single cistron or by a polar effect on a second cistron in the same operon. Introduction of a polarity suppressor caused no change in the overproduction of polysaccharide, in the enzymes of polysaccharide synthesis or in radiation sensitivity of the capR mutant. Thus mucoidy and radiation sensitivity resulting from capR (lon) mutations are both the consequences of impairment of the same cistron. The experiments demonstrate the advantage of the use of polarity suppressors (over conventional nonsense suppressors) in determining whether pleiotropic effects of a mutation are the result of polarity.     

Fidelity of Initiation of Protein Synthesis After Premature Chain Termination in Polarity Mutants

beta-Isopropylmalate dehydrogenase, the product of the second cistron of the leucine operon in Salmonella typhimurium, produced by strains bearing nonsense or frameshift mutations in the first cistron of the operon was shown to be homogeneous as judged by electrophoretic and immunological techniques. Amino terminal analyses suggest that the enzyme produced by the mutant strains is identical with the wild-type enzyme. This view is supported by the observation that a nonsense mutant strain beta-isopropylmalate dehydrogenase copurifies with the wild-type enzyme. The results suggest that the uncoupling of normal chain termination and reinitiation does not interfere with the fidelity of subsequent polypeptide chain initiation in a polycistronic messenger ribonucleic acid.     

Histidine regulation in Salmonella typhimurium. 8. Mutations of the hisT gene

The hisT gene, one of six genes in which mutation causes derepression of the histidine operon in Salmonella typhimurium, is shown to code for a protein that is not essential for the growth of the bacteria. This is indicated by the characterization of particular classes of mutations in the hisT gene: amber mutations, frame-shift mutations, and temperature-sensitive mutations that affect repression but not growth. In addition, the class of semilethal mutations was selected for but not found.     

“Self-Feeding” Strain of Salmonella typhimurium with a Mutation in the trpB Gene and Nutritional Requirements of trpA Gene Mutants

An indole-requiring (Ind(-)) mutant of Salmonella typhimurium, isolated from a culture of a leaky trpA mutant, was genetically analyzed by P22-mediated transduction. The mutation site giving the Ind(-) phenotype was shown to be in trpB, the second gene of the trp operon. A second mutation at this site resulted in change of nutritional requirement from indole to anthranilic acid (Anth(-)). This phenotype is normally associated with mutations in the first trp gene, trpA. However, the Anth(-) mutant also excreted anthranilic acid and showed "self-feeding" on unsupplemented media. Of two possible explanations for this aberrant phenotype, the first, that the trpB mutations may be in the "unusual" region, was dismissed on genetic evidence and on the biochemical evidence that an active anthranilate synthetase (AS) is produced. The alternative explanation, that the affected enzymatic activity, phosphoribosyl transferase, is unstable in vivo, but its AS component 2 activity is stable, is considered more probable.     

Analysis of promoter mutations in the histidine transport operon of Salmonella typhimurium: use of hybrid M13 bacteriophages for cloning, transformation, and sequencing.

Mutations that cause an increased level of expression of the histidine transport operon were isolated and characterized genetically. Five independently isolated promoter-up mutations were transferred to an M13 hybrid phage that carries the histidine transport operon, and their nucleotide sequences were determined. For all five mutations, the change was the same as the one previously determined for promoter-up mutation dhuA1: a C-to-T change in the Pribnow box rendered this region more homologous to the consensus sequence. Methods for enabling Salmonella typhimurium to support growth of M13 phage effectively and for easy transfer of chromosomal mutations onto the hybrid phage are presented.     

Two Mutations in the First Gene of the Histidine Operon of Salmonella typhimurium Affecting Control

Two strains with mutations in the first structural gene of the histidine operon of Salmonella typhimurium were characterized. (The first structural gene specifies the first enzyme of histidine biosynthesis, phosphoribosyltransferase, which is sensitive to feedback inhibition by histidine.) One mutation, hisG3934, results in a phosphoribosyltransferase which is no longer sensitive to feedback inhibition by histidine but is instead subject to inhibition by aspartic acid. The other mutation, hisG3935, allows the histidine operon to be partially repressed by several amino acids, including aspartic acid. Analysis of hisG3935 is consistent with the hypothesis that phosphoribosyltransferase is directly involved in the regulation of the histidine operon.     

Polar and Antipolar Mutants in the Tryptophan Operon of Salmonella typhimurium

Polar mutations in trpA, the first structural gene of the tryptophan operon of Salmonella typhimurium, have an uncoordinate effect on the expression of the distal genes, with trpB, the second gene, being more drastically affected than the last three. A number of these polar mutant strains grow very poorly on anthranilic acid-supplemented minimal medium. By selecting for more rapid growth in the presence of anthranilic acid, secondary mutant clones showing a correction of the polar effect were isolated. A few of these were analyzed and shown to contain deletions of various segments of the trpA gene. Ten randomly isolated deletion mutants missing various segments of the trp operon were analyzed for possible pleiotropic effects. Five of them showed a pleiotropic effect of some sort and five did not. Of those showing pleiotropic effects, one had lost the promotor-like elements necessary to initiate expression of the operon, three showed possible antipolar effects, and one showed both polar and antipolar effects simultaneously.     

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.     

Polarity Effects in the Hisg Gene of Salmonella Require a Site within the Coding Sequence

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.     

Characterization of SALMONELLA TYPHIMURIUM Mutants with Altered Glutamine Synthetase Activity

A number of glutamine auxotrophs of Salmonella typhimurium were isolated and characterized genetically. Three of the mutations appear to be closely linked and are complemented by episomes carrying the glnA region of Escherichia coli. The lesions in these strains are approximately 20% linked by P1 transduction with a mutation in the rha gene, but are unlinked to ilv. Another mutation causing glutamine auxotrophy in strain JB674 is genetically distinct from the others. Strain JB674 grown in glucose medium containing ammonia as the nitrogen source has reduced levels of glutamine synthetase that is more adenylylated than in the parent strain, suggesting that the enzyme can not be deadenylylated normally. The lesion causing glutamine auxotrophy in JB674 lies in the region corresponding to the glnB and glnE genes affecting glutamine synthetase modification in Klebsiella areogenes. Four Gln+ revertants of JB674 have glutamine synthetase activities 4 to 6 fold higher than normal. One mutation causing this increased enzyme synthesis has been shown by three-factor crosses with the glnA mutations to lie near or within the glnA gene.     

Polarity of amber mutations and suppressed amber mutations in the galactose operon of E. coli

Summary Amber mutants in the t gene of the galactose operon have been examined for polarity in the presence and absence of the suppressors su _I and su _yMel .In the absence of suppressors there is a gradient of polarity with the more polar mutations nearer the epimerase gene. This polarity is cis-dominant. Amber t mutants have raised epimerase levels but this effect is recessive. The operon is normally inducible in the presence of amber mutations. A double amber mutant had the polarity of the mutation nearest the epimerase end of the gene.In the presence of suppressors there is practically no gradient of polarity. This is in disagreement with the model proposed by Martin et al. (1966) and Yanofsky and Ito (1966). Modifications of this model to fit the present data are proposed.     

Polarity of amber mutations in ribosomal protein genes of Escherichia coli.

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.     

Mutants of Escherichia coli Lacking Endonuclease I, Ribonuclease I, or Ribonuclease II

To isolate mutants of Escherichia coli K-12 lacking endonuclease I activity (end), a method has been developed which detects, by differential methyl green staining, undegraded deoxyribonucleic acid (DNA) in colonies previously incubated in toluene. This procedure allows isolation of mutant strains in which DNA degradation is reduced. For half of these strains, this defect has been correlated with deficiencies of endonuclease I, ribonuclease I (rns), or ribonuclease II (rne) activities. The enzymatic deficiencies of the other strains remain unknown. An rne mutation is cotransducible with serA (which is located at 56 min on the genetic map). Most end mutations, called endA, are also cotransducible with serA and are located between serA and strA. One end mutation, called endB, is located between purE and trp (i.e., between 13 and 25 min on the genetic map).