Messenger Ribonucleic Acid Stability in Relaxed and Stringent Escherichia coli Starved for Methionine

During starvation for each of four amino acids, relaxed and stringent strains of Escherichia coli showed exponential decay of pulse-labeled unstable messenger ribonucleic acid (mRNA), with RNA degraded more slowly in the relaxed strain. An additional unique difference was observed during starvation for methionine: the relaxed strain showed non-exponential decay of mRNA, with a survival curve similar to that of an aging process.     

Stable Ribonucleic Acid Synthesis in Stringent (rel+) and Relaxed (rel−) Polyamine Auxotrophs of Escherichia coli K-12

The relationship of polyamines to stable ribonucleic acid (RNA) synthesis under conditions of amino acid withdrawal or chloramphenicol treatment was examined with the use of a closely related rel(+), rel(-) pair conditionally incapable of synthesizing putrescine. Under conditions of polyamine starvation, the cellular sperimidine level fell to one-third to one-half of the value observed in putrescine-supplemented cultures and putrescine became undetectable; cadaverine was synthesized by both strains, but the relaxed strain, MA 252, accumulated less cadaverine per cell than its stringent twin, MA 254. Upon amino acid withdrawal, the stringent strain remained stringent whether starved of or supplemented with polyamines. Similarly, the relaxed strain was capable of making RNA either with or without polyamine starvation. On the addition of chloramphenicol or upon amino acid withdrawal in the relaxed strain, supplementation with spermidine had no effect on the initial rate of RNA synthesis, although RNA accumulation was greater in the presence of added spermidine. Spermidine added at the conclusion of RNA synthesis prompted additional synthesis, although preincubation with spermidine again had no effect on the initial rate. All forms of stable RNA species were made with polyamine supplementation. The present data appear to rule out the possibility that polyamines are primary causative agents in stimulating RNA synthesis, but rather suggest an indirect or secondary role for spermidine in which the polyamines "stimulate" stable RNA synthesis probably by relieving RNA product inhibition of RNA synthesis.     

Control of Protein Synthesis in Escherichia coli: Analysis of an Energy Source Shift-Down

The energy source shift-down described in the preceding paper (Molin et al., J. Bacteriol. 131: 7-17, 1977) was used to study the effects of shift-down on protein synthesis. The overall rate of protein synthesis was reduced immediately, and to the same extent, in stringent and relaxed strains. The primary effect of the shift was a slowing down of the polypeptide chain growth rate, a finding not previously reported. In stringent strains the normal, preshift rate was reestablished within 2 to 3 min, whereas in relaxed strains the chain growth rate remained low for about 20 min before slowly returning to the normal value, which was reestablished some 50 to 60 min after the shift. Throughout this transition, the stability of messenger ribonucleic acid (mRNA) remained unchanged in both strains. We interpret these findings as evidence of the more rapid reduction of the mRNA pool in the stringent strain after shift-down: we believe that very soon after the shift, the stringent strain reduces its pool of mRNA and with it the number of ribosomes engaged in protein synthesis. In this manner the number of active ribosomes is adjusted to the availability of energy and carbon. The relaxed strain cannot rapidly reduce its mRNA pool, which thus remains large enough to engage a near-preshift number of ribosomes during a prolonged period; as a consequence its ribosomes must work at a reduced rate. The possibility that ppGpp is involved in the control of mRNA production is discussed. After shift-down, the initial part of beta-galactosidase (the auto-alpha fragment) was produced at a higher rate than complete beta-galactosidase in the relaxed strain, as expected when translation is impeded.     

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent (relA+)/relaxed (relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.     

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent (relA+) and relaxed (relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.     

Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.     

3H-thymidine incorporation following isoleucine limitation in stringent and relaxed controlled strains of Escherichia coli

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.     

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.     

Stringency and relaxation among the halobacteria.

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Different cyclic AMP requirements for induction of the arabinose and lactose operons of Escherichia coli

Valyl-tRNA deprivation causes a threefold reduction of the polysome content of stringent cells but not of relaxed cells. The polysomes of valyl-tRNA-deprived stringent and relaxed cells decay in the presence of rifampin at a rate very similar to that observed in growing cells.Polysome assembly and decay were studied in valyl-tRNA-deprived stringent and relaxed strains after first causing the pre-existing polysomes to be converted to monosomes by glucose starvation. The capacity for polysome assembly is normal in relaxed cells and is reduced by a factor of three in stringent cells. The polysomes which reassemble in glucose-starved cells also decay in the presence of rifampin at a rate similar to that of the polysomes of growing cells. The polysomes which assemble in relaxed cells are potentially functionally competent, as shown by their ability to incorporate amino acids in an in vitro proteinsynthesizing system. Valyl-tRNA deprivation causes an intense shift in the polysome size distribution in stringent cells, but only a moderate shift in relaxed cells. A model for the control of the polysome level in amino acid-starved cells, based on these observations, is presented here.     

Conformational changes in bacterial polysomes induced by amino acid starvation

The effects of amino acid starvation on polysome conformation were analyzed comparatively in stringent (relA+) and relaxed (relA) bacteria by measuring the accessibility in vitro of ribosomal proteins to reductive methylation. In polysomes of stringent cells, the conformational state of two proteins (L13 and L29) appeared significantly changed by starvation. In polysomes isolated from relaxed mutants, the accessibility of five proteins (L5, L13, L29, L31 and L32) was found modified.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.     

Control of Nucleotide Metabolism and Ribosomal Ribonucleic Acid Synthesis During Nitrogen Starvation of Escherichia coli

Ribosomal ribonucleic acid (RNA) synthesis and ribonucleoside triphosphate metabolism were studied in cultures of Escherichia coli subjected to starvation for inorganic nitrogen. In a strain that was under stringent control, a 50-fold reduction in the formation of both 16S and 23S RNA was accompanied by a severe restriction on nucleotide biosynthesis. These inhibitions were relieved in part by incubating the starved cells with amino acids. This result suggests that regulation by the functional RNA control (RC) gene is involved in the effect. This suggestion was confirmed by showing that the effector of the stringent response, guanosine-5'-diphosphate-2'- or 3'-diphosphate ((pp)G(pp)), accumulated at the onset of starvation and disappeared immediately when the amino acids were added. Ribosomal RNA synthesis was severely restricted and the same nucleotide, (pp)G(pp), accumulated at the onset of nitrogen starvation of a relaxed mutant too. These findings suggest that a control mechanism other than the one provided by the functional rel gene might operate to regulate RNA synthesis and that this mechanism is expressed through the synthesis of (pp)G(pp).     

Synthesis, no synthesis, or synthesis and degradation of ribosomal ribonucleic acid in various Escherichia coli strains starved for an amino acid

A standard stringent strain of Escherichia coli makes little or no ribosomal ribonucleic acid (RNA) during starvation for an essential amino acid, whereas the isogenic relaxed strain makes both ribosomal and messenger RNA. A third class of strains was found which continues to make ribosomal RNA during starvation, but the RNA made is apparently unstable. There is little accumulation of RNA in the third class of strains, and few complete newly formed chains of (3)H-ribosomal RNA are observed in sedimentation analyses, even after long labeling times.     

Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

We have investigated the changes in the guanosine 5'-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5'-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5'-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5'-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.     

Polyamine regulation of stringent control in a polyamine-auxotrophic strain of Escherichia coli.

Escherichia coli BGA8 , a mutant unable to synthesize putrescine, behaves as stringent or relaxed according to the presence or absence of polyamine, respectively, in the culture medium. The relaxed synthesis of RNA can be reverted back to stringent by addition of putrescine or spermidine. The stringent response depends on the concentration of the polyamine in the culture medium. The formation of guanosine 3'-diphosphate 5'-diphosphate elicited by amino acid starvation is stimulated at least 40-fold in putrescine-supplemented bacteria and only about 2-fold in putrescine-depleted cells.