Excssive DNA synthesis inLactobacillus acidophilus during amino acid starvation

Replication of the bacterial chromosome was studied in two substrains ofLactobacillus acidophilus R-26 during amino acid starvation. According to the hypothesis of Maaløe and Hanawalt (1961), already initiated DNA replication cycles are completed under such conditions, with a corresponding 40% increase in the DNA content; new cycles cannot be initiated in the absence of proteosynthesis. Our findings are considerably at variance with this hypothesis. It was found that the course of DNA synthesis and the size of DNA increments during amino acid starvation were influenced by some low molecular weight substances, in particular by deoxyadenylate and spermidine. In the presence of these substances in media without the essential amino acids, prolonged DNA synthesis accompanied by large DNA increments was observed, suggesting that new DNA replication cycles were initiated. The possibility that deoxyadenylate and spermidine influence the regulation of synthesis of the bacterial chromosome is discussed.     

Stringent control of RNA synthesis inLactobacillus acidophilus

The amino acid regulation of RNA synthesis inLactobacillus acidophilus was studied and found to be of stringent character. The synthesis of RNA was inhibited in the absence of essential amino acids in the medium, this inhibition being released by chloramphenicol or chlortetracycline. The RNA synthesized in the presence of the above inhibitors was not stable. The results do not support the hypothesis that the release of RNA synthesis by chloramphenicol is due to an increased pool of free amino acids, in consequence to the inhibition of protein synthesis. Chloramphenicol removed the inhibition of RNA synthesis at the same rate as the amino acids themselves. The pool of free leucine or histidine decreased to 60% in the absence of exogenous amino acids and it was not raised on adding chloramphenicol. The results are in agreement with the assumption that the synthesis of ribosomal RNA in bacteria is controlled by the equilibrium between polysomes and free ribosomes. Further, the results point to a possible limiting role of proteins in the regulation of ribosomal RNA synthesis.     

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: Stable DNA replication

After inhibiting DNA synthesis in Escherichia coli, repeated cycles of chromosome replication can occur in the absence of protein synthesis. This “stable” replication requires the products of all of the known dna genes.Stable replication results from inhibiting DNA synthesis by treatment with naladixic acid, cytosine arabinoside or hydroxyurea; or by placing dnaB, dnaE or dnaG mutants at non-permissive temperatures. It also follows a “shift-up” into rich medium in which RNA and protein are synthesized more rapidly than DNA. Paradoxically, stable replication is induced also by treatment with concentrations of streptolydigin which do not inhibit DNA replication but temporarily and partially inhibit RNA and protein synthesis. During all of these treatments, some protein synthesis must occur.Stable replication is not immediately expressed after a short period of thymine starvation or streptolydigin treatment, but requires a subsequent period of protein synthesis. Once established, however, the stable replication state is permanent and will persist in the absence of protein synthesis or during normal growth.After stable replication has been determined by a period of DNA inhibition, it is possible to inactivate replication by heating dnaA, B, C, E and G temperature-sensitive mutants. However, resynthesis of these gene products in the presence of thymine and at the permissive temperature restores stable replication activity. Since restoration of activity can occur under normal growth conditions which do not induce stable replication, it was concluded that the dnaA, B, C, E and G gene products do not directly determine the stabilized character of the replication fork.A model is presented which attempts to explain the ability of different treatments to induce stable replication.     

Noncoordinate control of RNA, lipopolysaccharide, and phospholipid syntheses during amino acid starvation in stringent and relaxed strains of Escherichia coli.

The syntheses of RNA, lipopolysaccharides, and phospholipids were measured simultaneously in stringent and relaxed cells of Escherichia coli during normal growth or starvation for amino acids. The synthesis of all these molecules was inhibited by amino acid starvation, but the reduction in synthesis was not coordinated.     

Replication of lambda plasmid in amino acid-starved strains of Escherichia coli.

We found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant of Escherichia coli K-12, whereas is inhibited in its wild-type stringent partner. Replication of lambda plasmid in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. The replication also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the signal for the stringent response, on RNA polymerase.     

Effect of Rifampicin and Chloramphenicol on Progeny Single-stranded DNA Synthesis of Bacteriophage ϕX174

Neither bacteriophage ?X174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ?X174 DNA synthesis started.     

Positioning of replicated chromosomes in Escherichia coli.

The positioning of replicated chromosomes at one-fourth and three-fourths of the cell length was inhibited when protein synthesis was inhibited by chloramphenicol or rifampin or by starvation for amino acids. Under these conditions, the progress of chromosome replication continued and replicated chromosomes were located close to each other as one nucleoid mass at midcell. Cells which already had two separate daughter chromosomes located at the cell quarters divided into two daughter cells under these conditions. When protein synthesis resumed, daughter chromosomes moved from midcell to the cell quarters, respectively, before any detectable increase in cell length was observed. The chromosome positioning occurred even under inhibition of the initiation of chromosome replication and under inactivation of DNA gyrase. The chromosome positioning presumably requires new synthesis of a particular protein(s) or translation itself.     

Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after x-irradiation

Billen, Daniel (The University of Texas, Houston), and Roger Hewitt. Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after X irradiation. J. Bacteriol. 90:1218-1225. 1965.-A study was made of the effect of amino acid deprivation or chloramphenicol on the character of postirradiation deoxyribonucleic acid (DNA) replication in bacteria with the use of radioisotopes and 5-bromouracil as a density label. CsCl density-gradient studies of DNA showed that postirradiation incubation of amino acid-requiring Escherichia coli in an amino acid-free medium interfered with continued linear chromosomal replication. In the presence of the required amino acids, linear chromosomal replication was shown to resume. Addition of chloramphenicol was found to prevent this resumption. Deletion of the required amino acids or the presence of chloramphenicol in a fully supplemented medium allowed the detection of altered DNA synthesis in bacteria at X-ray doses as low as 500 r. The character of the limited DNA made in the presence of the density label after irradiation is described. The results are interpreted as showing that the synthesis of a protein(s) is required for restoration of linear chromosomal replication in the irradiated cells.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Ribonucleic Acid Regulation in Amino Acid-Limited Cultures of Escherichia coli Grown in a Chemostat

The regulation of ribonucleic acid (RNA) synthesis was examined in cultures of bacteria whose growth was limited in the chemostat by the supply of a required amino acid. Strains possessing the relaxed (relA) mutation accumulated excess RNA (relative to protein) at low growth rates when growth was limited by arginine, histidine, or cysteine but not when limited by methionine. In contrast, stringent (relA(+)) strains maintained a constant RNA/protein ratio with decreasing growth rate regardless of the amino acid used to limit growth. The presence of excess RNA in relaxed strains was accompanied by an absence of increase in RNA production upon addition of chloramphenicol, a lag upon shift-up in growth by addition of excess of the limiting amino acid, and a decreased rate of production of beta-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50S and 30S ribosomal subunits. Microscope examination of the relaxed strains during histidine-, arginine-, or cysteine-limited growth in the chemostat showed them to be 10 to 20 times longer in size than the stringent strains. Also, cell density was reduced to one-tenth when the increased size was observed. An analysis of the amount of ppGpp present in all slow-growing amino acid-limited cultures (relaxed and stringent) demonstrated that only basal levels of ppGpp were made. These data are consistent with the hypothesis that when growth is limited in the chemostat by an initiation event in protein synthesis, i.e., limited methionine, RNA regulation occurs in relaxed as well as stringent strains. Also, when other amino acids are limiting in concentration during translation, errors occur in relaxed strains, resulting in misread proteins.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

Effect of Growth Conditions on the Formation of the Relaxation Complex of Supercoiled ColE1 Deoxyribonucleic Acid and Protein in Escherichia coli

Colicinogenic factor E1 (ColE1) is present in Escherichia coli strain JC411 (ColE1) cells to the extent of about 24 copies per cell. This number does not appear to vary in situations which give rise to twofold differences in the amount of chromosomal deoxyribonucleic acid (DNA) present per cell. If cells are grown in the absence of glucose, approximately 80% of the ColE1 molecules can be isolated as strand-specific DNA-protein relaxation complexes. When glucose is present in the medium, only about 30% of the plasmid molecules can be isolated as relaxation complexes. Medium shift experiments in which glucose was removed from the medium indicate that within 15 min after the shift the majority (>60%) of the plasmid can be isolated as relaxation complex. This rapid shift to the complexed state is accompanied by a two- to threefold increase in the rate of plasmid replication. The burst of replication and the shift to the complexed state are both inhibited by the presence of chloramphenicol. Inhibition of protein synthesis in log cultures by the addition of chloramphenicol or amino acid starvation allows ColE1 DNA to continue replicating long after chromosomal replication has ceased. Under these conditions, noncomplexed plasmid DNA accumulates while the amount of DNA that can be isolated in the complexed state remains constant at the level that existed prior to treatment. In the presence of chloramphenicol, there appears to be a random dissociation and association of ColE1 DNA and “relaxation protein” during or between rounds of replication.     

Amino acid deprivation induces synthesis of four proteins in chick embryo cells

Amino acid deprivation of chick embryo cells enhances the synthesis of four proteins whose molecular weights are approx. 89000, 73000, 35000 and 27000. This enhancement, which is seen in medium completely free of amino acids, can be prevented by the addition of any single amino acid. Furthermore, in the absence of amino acids in the medium, DNA and RNA synthesis is markedly inhibited, an effect which is similarly prevented by the addition of single amino acids. These new proteins synthesized in the amino acid-free medium co-migrate on one-dimensional gels with the ‘stress proteins’ induced by a variety of agents such as heavy metals, sulfhydryl reagents, heat shock, and amino acid analogues.     

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.     

PHA stimulation of human lymphocytes during amino acid deprivation. Protein,RNA and DNA synthesis

Resting lymphocytes are in the G₀ phase of the cell cycle. Upon activation by PHA, they progress into G₁ with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine-deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine). Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan- or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation. Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.