Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli

Ben-Hamida, Fakher (Washington University School of Medicine, St. Louis, Mo.), and David Schlessinger. Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli. J. Bacteriol. 90:1611-1616. 1965.-Synthesis of beta-galactosidase stops within several minutes when preinduced, permeaseless cultures are diluted into medium containing 40 mug/ml of 5-fluorouracil (5-FU) but no inducer. However, if inducer (isopropylthiogalactoside) is left in the medium, enzyme formation in the presence of 5-FU continues for at least 11 min. Thus, inducer may increase the differential metabolic stability of the corresponding messenger ribonucleic acid (RNA; defined as the capacity to produce measurable enzyme) in inducible strains. However, such an interpretation requires that 5-FU rapidly arrest the further synthesis of messenger RNA competent to form active enzyme. C(14)-5-FU, like uracil, does appear to enter cells without measurable lag, saturating the pool of uracil nucleotides, and thereby the messenger RNA being formed, within several minutes. That 5-FU acts very quickly is also supported by the similar continuation of enzyme synthesis in the presence of inducer and antibiotics (actinomycin D and proflavine) which shut off all RNA synthesis, as well as by the response to 5-FU of enzyme synthesis in various constitutive mutants.     

Influence of Polyamine Limitation on the Chain Growth Rates of β-Galactosidase and of Its Messenger Ribonucleic Acid

The rates of elongation of beta-galactosidase and its messenger ribonucleic acid (RNA) were estimated in a polyamine-deficient mutant of Escherichia coli through an analysis of the kinetics of enzyme induction. The chain growth of beta-galactosidase was calculated from the time required after the appearance of an amino terminal fragment of 60 amino acids (auto-alpha) until completed enzyme began to accumulate. The elongation rate of beta-galactosidase messenger RNA was estimated from the time after induction at which streptolydigen-resistant, enzyme-forming capacity first appeared. Upon polyamine starvation, the rate of polypeptide elongation slowed from 17 to 10 amino acids per s and the messenger RNA elongation rate decreased from 47 to 30 nucleotides per s. These reductions in polymerization rates were proportional to the decrease in cellular growth rate produced by polyamine starvation. It was concluded that, although it is quite unlikely that polyamine levels are involved in regulation of cell growth, they may be acting as cofactors in the synthesis of RNA or protein, or both.     

Transient repression of catabolite-sensitive enzyme synthesis elicited by 2,4-dinitrophenol.

Transient inhibition of catabolic enzyme synthesis in Escherichia coli occurred when a low concentration of 2,4-dinitrophenol (DNP) was simultaneously added with inducer. Using mutant strains defective for gamma-gene product or constitutive for lac enzymes, it was found that the inhibition is not due to the exclusion of inducer by uncoupling. The addition of cyclic adenosine 3',5'-monophosphate overcame repression. The components of the lac operon coordinately responded to DNP inhibition. From deoxyribonucleic acid-ribonucleic acid hybridization experiments, it was found that the inhibition of beta-galactosidase induction occurred at the level of messenger ribonucleic acid synthesis specific for the lac operon. It seems probable that DNP represses induction in a similar manner to that of transient repression observed upon the addition of glucose. Furthermore, it was found that transient repression disappeared if cells were preincubated with DNP before induction. This indicates that new contact of cells with DNP is obligatory for transient repression. From these results, it is suggested that the cell membrane may be responsible for regulation of catabolite-sensitive enzyme synthesis.     

Synthesis of inducible enzyme in Escherichia coli recovering from prolonged energy starvation.

A marked breakdown of ribosomes and rRNA occurs in Escherichia coli cells during prolonged deprivation of a carbon source (energy starvation). In E. coli recovering from energy starvation: (a) synthesis of RNA started immediately, total protein synthesis showed a delay of 5 to 10 minutes; (b) beta-galactosidase, tryptophanase and serine deaminase could not be induced in the first 50--70 min; (c) a lag of 60 min in the synthesis of beta-galactosidase was observed in a lac constitutive mutant of E. coli; synthesis of the constitutive enzyme malate dehydrogenase did not shown any delay. RNA synthesized in the early stages of recovery contained a higher percentage of low molecular weight molecules than RNA synthesized after 70 min of recovery or during exponential growth. Messenger RNA specific for beta-galactosidase was not synthesized for the first 50--60 min of recovery even when the specific inducer was added to the cultures.     

Role of lac genes in induction of beta-galactosidase synthesis by galactose

Strain BL1003, a lacO mutant, synthesizes beta-galactosidase constitutively at a low rate. The enzyme is further inducible by d-galactose to the same differential rate as is seen in the presence of an optimal concentration of thiomethylgalactoside. lacY Mutants derived from strain BL1003 are not inducible by galactose, although they synthesize beta-galactosidase at the low constitutive rate characteristic of the parent. Galactose is a weak inducer of beta-galactosidase synthesis in wild-type Escherichia coli K-12, but it is more effective when the wild type has been preinduced with isopropyl-beta-d-thiogalactoside. Nevertheless, the rise in the differential rate of synthesis in response to galactose in a preinduced wild-type culture is much lower than in strain BL1003. Thus, two factors are involved in the induction of strain BL1003 by galactose: the mutant operator and the constitutive permease. The operator has an altered sensitivity to the i product-galactose complex. The low constitutive level of permease enabled the cells, at the high concentrations of galactose used (5 x 10(-2)m), to maintain a sufficient internal concentration for further induction.     

Effect of Infection with Ribonucleic Acid Bacteriophage R23 on the Inducible Synthesis of β-Galactosidase in Escherichia coli

Infection by ribonucleic acid (RNA) bacteriophage R23 inhibited the synthesis of beta-galactosidase in Escherichia coli. The inhibition, although not complete, was apparent shortly after infection and was maximal after the first 20 min of infection. R23 diminished the beta-galactosidase-synthesizing capacity when inducer was added after phage infection, but not when infection followed inducer removal. These findings suggested that the primary effect of R23 on enzyme-forming capacity was limitation of synthesis of enzyme-specific messenger RNA. Studies with ultraviolet irradiated phage and amber mutants of R23 indicated that the inhibitory process could be separated into two phases. Early inhibition did not require the expression of the viral genome, whereas late inhibition required the expression of the viral RNA synthetase cistron.     

Regulation of Penicillinase Synthesis: Evidence for a Unified Model

The kinetics of penicillinase induction in Bacillus cereus 569 was investigated. An increase in the rate of penicillinase synthesis was demonstrated within 30 sec of the addition of inducer (benzylpenicillin); however, the maximum induced rate of penicillinase synthesis was not attained until at least 30 min after the addition of inducer. In contrast to earlier claims, a quantitative estimate showed that the penicillinase messenger ribonucleic acid (mRNA) half-life is approximately 2 min. These findings strongly suggest that the rate of synthesis of penicillinase mRNA increases continuously during most of the 30-min latent period. A model for the regulation of penicillinase synthesis in three gram-positive organisms is presented which is consistent with a nondiffusible inducer, a short-lived mRNA, a relatively long latent period (i.e., an apparently slow inactivation of penicillinase repressor), and the existence of at least two regulatory genes.     

Molecular events associated with induction of arginase in Saccharomyces cerevisiae.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.     

Lifetime of messenger ribonucleic acid for penicillinase synthesis in several strains of bacilli

1. Previous studies of penicillinase synthesis in Bacillus licheniformis showed that enzyme synthesis after the addition of actinomycin continues for far longer in the constitutive mutant 749/C than in the parental inducible strain (Yudkin, 1966). This result was interpreted as indicating a difference in the lifetime of specific messenger RNA in the two strains. Other bacilli have now been examined in an attempt to see whether this difference is general. 2. There was no difference in the lifetime of messenger RNA for penicillinase synthesis between an inducible and a constitutive strain of Bacillus cereus. 3. Three freshly isolated constitutive mutants of B. licheniformis also had short-lived messenger RNA, like their inducible parent. 4. A reinvestigation of mutant 749/C confirmed the original finding that, on treatment with actinomycin, it continued to synthesize penicillinase far longer than did its parent. 5. An inducible revertant of mutant 749/C was indistinguishable from the original inducible strain, and appeared to have lost both constitutivity and long-lived messenger RNA in the back mutation.     

Discontinuous synthesis of lac messenger ribonucleic acid in a mutant of Escherichia coli with a new lac promoter.

A mutant of Escherichia coli with a new promoter for the lac operon exhibits dramatic discontinuities in the synthesis of lac messenger ribonucleic acid after induction. These discontinuities immediately precede similar discontinuities in the synthesis of beta-galactosidase. The discontinuous synthesis of beta-galactosidase persists after addition of rifampin.     

Beta-Galactosidase messenger RNA made during recovery from inhibition of protein synthesis is not translated.

Bacteria that accumulate RNA in the course of inhibition of protein synthesis are impaired in their ability to synthesize beta-galactosidase during subsequent recovery. By contrast, constitutive enzyme synthesis in recovering cells is normal. Even though no beta-galactosidase is made during recovery from this inhibition, a substantial quantity of beta-galactosidase mRNA (as determined by DNA-RNA hybridization) is made. The beta-galactosidase mRNA made in vivo is functional in vitro. It is capable of directing the in vitro synthesis of a portion of the NH2-terminal beta-galactosidase molecule (in the alpha portion of the molecule). However, this protein is not made in vitro. It is concluded that the beta-galactosidase mRNA that is made during recovery from protein synthesis inhibition, although apparently at least partly normally transcribed in vivo and functional in vitro cannot be translated under these conditions in vivo.     

Transient Repression of the lac Operon

Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression.     

Export of extracellular levansucrase by Bacillus subtilis: inhibition by cerulenin and quinacrine.

Bacillus subtilis B secretes an inducible, extracellular enzyme, levansucrase. Inhibition studies were undertaken to investigate the possible mechanism of release of this enzyme. The antibiotic cerulenin, at a concentration of 10 micrograms/ml, totally inhibited de novo lipid synthesis in B. subtilis B for at least 1 h, while only slightly reducing protein and RNA synthesis. At this concentration cerulenin, added concomitantly with the inducer sucrose, prevented the release of levansucrase for at least 150 min. This was not due to the prevention of inducer uptake by the cells. The release of the enzyme was also independent of cell division. In B. subtilis 1007 the induction of beta-galactosidase by 5 mM lactose was not prevented by cerulenin. Preliminary evidence indicated the association of a lipid moiety with the enzyme as it passes through the cytoplasmic membrane. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing protease of Bacillus licheniformis, inhibited levansucrase release from B. subtilis B, but had no effect on lipid synthesis.