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1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

The lon(-) mutation is responsible for a defect in cell division of Escherichia coli lightly irradiated with ultraviolet light (UV). These lon(-) mutants can be isolated readily by a procedure described here. Physiological studies were performed with the objective of determining the role of the lon gene. Unirradiated lon(-) mutants grow normally, except that a correlation of this mutation with capsule formation has been noted previously. These two properties can be separated, however. After irradiation, lon(-) grows as long filaments because septum formation is prevented. The filaments eventually lyse. Mass increase and deoxyribonucleic acid and enzyme synthesis appear to proceed normally. Thus, the lesion produced by UV appears to be highly specific. In bacteria that carry both genes (merozygotes), lon(+) is dominant to lon(-). Septum formation is restored to irradiated lon(-) bacteria by introduction of lon(+) by conjugation. Also, normal growth can be restored by nutritional variations. It is concluded that lon(+) is able to nullify the effects of the UV lesion under conditions where lon(-) cannot. Possibly, capsule precursors that can accumulate in the latter are responsible for the difference because they interfere with repair of the UV lesion.

Both cyclic AMP and a specific inducer acting in concert are required for the synthesis of many inducible enzymes in E. coli. Little enzyme is made in the absence of either. In contrast to the specific inducers which stimulate the synthesis only of the proteins required for their metabolism, cyclic AMP controls the synthesis of many proteins. Glucose and certain other carbohydrates decrease the differential rate of synthesis of inducible enzymes by lowering cyclic AMP concentrations. In the lac operon, cyclic AMP acts at the promoter site to facilitate initiation of transcription. This action requires another protein, the cyclic AMP receptor protein. The nucleotide stimulates tryptophanase synthesis at a translational level. The action of cyclic AMP in E. coli may serve as a model to understand its action on transcriptional and translational processes in eukaryotes.

When Neurospora mycelium is transferred from a medium containing sucrose to one containing acetate as sole source of carbon, a preferential synthesis of many Krebs cycle, glyoxylate cycle, and associated enzymes occurs. Respiration was inhibited during preferential enzyme synthesis in the following ways. (i) The amount of aeration (shaking) was reduced, (ii) cyanide was added to the culture, (iii) the carbon source, acetate, was removed, (iv) a mutant strain was starved of its Krebs cycle intermediates, and (v) respiration was inhibited by mutation. The effect of this respiratory inhibition on the synthesis of a number of enzymes was measured. It was found that the synthesis of nicotinamide adenine dinucleotide (NAD)-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase was significantly less inhibited under conditions of respiratory inhibition than was the synthesis of Krebs cycle, glyoxylate cycle, and most other cell proteins synthesized during the adaptation period. This differential inhibition of enzyme synthesis was almost certainly not due to differential repression by regulatory metabolic end product effectors. Inhibition of mitochondrial respiration under these conditions most likely results in a limitation of the energy supply of the cell. Thus, it is suggested that the inhibition of synthesis of most proteins after inhibition of mitochondrial respiration results from a lack of energy in a utilizable form. Possible reasons to account for the relative insensitivity of NAD-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase to inhibition under these conditions are discussed.

Synthesis of the secondary metabolite, actinomycin, and the enzyme, phenoxazinone synthase, involved in the biosynthesis of the antibiotic, were shown to be under severe catabolite repression by glucose. Of a variety of hexoses and carbon compounds examined, glucose, and to a lesser extent, mannose, proved to be the most repressive for enzyme synthesis. The repression by glucose was most evident before production of the antibiotic. In a chemically defined medium suitable for actinomycin production, synthesis of phenoxazinone synthase began at the time the glucose (0.1%) supply was depleted. Soon after, antibiotic synthesis was initiated. Galactose, the major carbon source for growth and antibiotic synthesis, was not utilized until the glucose was consumed. Generally, carbon compounds which supported a rapid rate of growth were most effective in producing catabolite repression.

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.

Megraw, Robert E. (Iowa State University, Ames), and Russell J. Beers. Glyoxylate metabolism in growth and sporulation of Bacillus cereus. J. Bacteriol. 87:1087-1093. 1964.-Isocitrate lyase and malate synthetase were found in cell-free extracts of Bacillus cereus T. The patterns of synthesis of enzymes of the glyoxylic acid cycle were dependent upon the medium in which the organism was grown. Cells grown in acetate or in an acetate precursor, such as glucose, produced enzymes of the glyoxylic acid cycle in greatly diminished quantities, as compared with cells grown in media containing glutamate or yeast extract as principal carbon sources. Glutamate-grown cells had high isocitrate lyase activity but very low malate synthetase activity. Glyoxylate produced in this situation is metabolized by alternate pathways: conversion to tartronic semialdehyde and the latter to glyceric acid, thus providing evidence for a glycerate pathway; and reduction to glycolate (the reverse of this reaction was present at a low rate). Enzymatic activity of the glyoxylic acid cycle declines at the point where sporogenesis begins, indicating a metabolic shift for the synthesis of spore material.

Experiments were carried out to determine whether, during outgrowth of bacterial spores, deoxyribonucleic acid (DNA) replication provided the basis by which ordered transcription was controlled. During outgrowth, significant DNA synthesis does not occur until just prior to the onset of cell division. However, incorporation of radioactive DNA precursors into DNA is observed within 5 to 10 min after the initiation of germination. By employing a thymine-requiring auxotroph and (3)H-bromodeoxyuridine, this incorporation appears to be a result of DNA replication and not repair synthesis. For the following reasons it was concluded that, during outgrowth, transcriptional processes were not ordered by DNA replication. (i) In a thymine auxotroph, thymine addition did not alter the periodicity of induced alpha-glucosidase and histidase synthesis during outgrowth. (ii) DNA synthesis was inhibited 80% by 5-fluoro-2'-deoxyuridine (FUdR), and, after a 5-min lag, completely by mitomycin C, but these inhibitors exerted a differential effect on induced histidase synthesis. Enzyme synthesis was insensitive to FUdR but was inhibited by mitomycin C, presumably as a result of cross-linking of the complementary DNA strands.

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.

A study was made of how the catabolism of a carbon and energy source is affected by the biosynthetic demands of growing bacterial cells. Cultures of Aerobacter aerogenes in l-histidine medium were grown in a chemostat at rates determined by the supply of either sulfate or a required amino acid, l-arginine. It was discovered that the rate at which these cells grow under a biosynthetic restriction determines both the rate and the pattern of histidine degradation. (i) Histidine catabolism is partially coupled to the growth rate. This coupling is achieved by catabolite repression of histidase (histidine ammonia lyase; EC 4.3.1.3.), and also by a slightly decreased in vivo function of this enzyme at low growth rates. (ii) The looseness of the coupling results in a direct relationship between growth rate and growth yield, and possibly is correlated with an altered pattern of carbon flow from histidine. (iii) Sudden decreases in growth rate cause total repression of histidase synthesis for substantial periods of time. (iv) Sudden release of biosynthetic restriction leads rapidly to an increase in the functioning of the cells' complement of histidase, an increase in the rate of synthesis of this enzyme, and an increase in the growth yield from histidine.

A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.

l-Threonine deaminase (l-threonine dehydratase [deaminating], EC 4.2.2.16) has been shown to be involved in the regulation of three of the enzymes of isoleucine-valine biosynthesis in yeast. Mutations affecting the affinity of the enzyme for isoleucine also affected the repression of acetohydroxyacid synthase, dihydroxyacid dehydrase, and reductoisomerase. The data indicate that isoleucine must be bound for effective repression of these enzymes to take place. In a strain with a nonsense mutation midway in liv 1, the gene for threonine deaminase, starvation for isoleucine or valine did not lead to derepression of the three enzymes; starvation for leucine did. The effect of the nonsense mutation is recessive; it is tentatively concluded, therefore, that intact threonine deaminase is required for derepression by two of the effectors for multivalent repression, but not by the third. A model is presented which proposes that a regulatory species of leu tRNA(leu) is the key intermediate for repression and that threonine deaminase is a positive element, regulating the available pool of charged leu tRNA by binding it.

1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.