Growth and Metabolic Activities of Heat Treated Staphylococcus aureus

Staphylococcus aureus cells which had been heated at 50 or 60° were transferred to various growth media and intracellular ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and amino acids were measured during the lag phase of growth. The duration of the lag phase depended on the temperature to which the cultures had been subjected, and was longest following storage at 60°. RNA synthesis occurred almost immediately on placing treated cells in a growth medium, but at a slower rate than with unheated cells. Variation in the composition of the metabolic pool of heated cells occurred during the early lag phase and may be as a result of damage to the cytoplasmic membrane with resulting loss of permeability control.     

An immediate and steep increase in ATP concentration in response to reduced turgor pressure in Escherichia coli B

Osmotic treatment with sodium chloride of Escherichia coli B growing in the logarithmic phase induced an immediate increase in ATP concentration in response to increased concentrations of added solute in its growth medium in the first 10 min of the addition. After that, ATP concentration decreased gradually. Sodium arsenate and potassium fluoride almost abolished the ATP increase. The time course of the increase was quite different from that of cells treated with inhibitors of protein synthesis. The osmotic treatment did not decrease the viability of cells. In addition, there was no degradation of RNA by 5 min after sodium chloride addition, and, further, the lag time of ATP increase was extended by an inhibitor of nucleotide synthesis. These results indicated that a major fraction of the stress-increased ATP resulted from de novo synthesis, and that it was mainly dependent upon the reaction of substrate-level phosphorylation, which is regulated by turgor pressure.     

Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth

When a culture of Escherichia coli ML30 growing exponentially at 37 C in a glucose minimal medium was shifted abruptly to 10 C, growth decreased for about 4.5 hr. There was no net synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. The cells, however, respired at a rate characteristic of cells growing in the steady state at 10 C and were able to accumulate alpha-methyl-d-glucoside. When growth recommenced at 10 C, protein synthesis started at 4 hr, RNA synthesis, with a burst at 6 hr, and DNA synthesis, with a burst at 7 hr. One synchronous division occurred at about 11 hr after shifting to 10 C. There was no alteration in the steady-state RNA to protein ratio. The results are discussed in relation to other reported effects of shifts in environmental conditions. The lag at 10 C was dependent on prior conditions of growth at 37 C. Growth at 37 C under conditions giving catabolite repression were necessary for the lag to be established on shifting to 10 C.     

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.     

Membrane damage in abortive infections of colicin Ib-containing Escherichia coli by bacteriophage T5.

Strains of Escherichia coli K-12 containing the colicin Ib (Col Ib) factor did not produce progeny phage when infected by T5 bacteriophage. The cells were killed but did not lyse. If sodium dodecyl sulfate (SDS) was added to T5-infected E. coli (Col Ib), lysis occurred prematurely, but no phage were produced. SDS had no effect on infected cells that did not contain the Col Ib factor or on uninfected cells with or without the Col Ib factor. Cells that contained a mutant Col Ib factor that allowed phage production were not prematurely lysed after infection in the presence of SDS. When the Col Ib-containing cells were infected, protein and RNA synthesis stopped at about 10 min postinfection, and the cells released abnormal amounts of 32P-containing material, ATP, and beta-galactosidase into the medium. They also became inhibited in their ability to accumulate thiomethyl-beta-D-galactopyranoside and to utilize glycerol. Two alternative hypotheses are presented to explain these results.     

Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation.

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant.     

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.     

Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were alpha-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr>25000). Over a 2h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional 'holes' of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and nonconfluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional 'holes' of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Inhibition of bacterial growth by metal salts. A survey of effects on the synthesis of ribonucleic acid and protein

Inhibition of the growth of Escherichia coli M.R.E. 600 by six different metal salts was accompanied by a greater decrease in the synthesis of RNA than in that of protein. The action of cobalt chloride was exceptional; inhibited cells made an excess of RNA to an extent depending on the concentration of Co(2+), the time of incubation and the concentration of Mg(2+) in the medium. Preferential synthesis of RNA in the presence of cobalt chloride was not confined to E. coli but occurred to various extents in some, but not all, of the other micro-organisms that were tested. Possible reasons for the special effect of Co(2+) are discussed.     

Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6-8 h after an initial 2-3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.     

Further characterization of the effect of bacterial lipopolysaccharide preparations on cyclic GMP levels: the importance of macromolecular synthesis

Bacterial lipopolysaccharides (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver cells without affecting the concentration of cAMP. This effect is produced by very small (1 ng) amounts of LPS and is both dose and time dependent. The time dependence is characterized by an initial lag period of 60-120 min followed by a rapid, persistent increase in cGMP levels. Since this time course suggests that synthesis of an intermediate might play an important role in the cGMP elevation, a series of experiments was done to evaluate the effect of LPS on DNA, RNA, and protein (macromolecular) synthesis. LPS did not measurably effect total macromolecular synthesis. However, inhibitors of RNA and protein synthesis markedly reduced cGMP levels in LPS-treated cells, whereas inhibition of DNA synthesis did not. Addition of sodium nitroprusside to control and inhibitor-treated cultures produced large equivalent increases of cGMP levels in both cases, indicating that the cells present were fully capable of responding to a stimulus of guanylate cyclase. Taken together, this data suggests that expression of the LPS-cGMP response in fetal liver cells is dependent on synthesis of an intermediary protein(s) during the lag phase.     

Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

The synthesis of ribonucleic acid (RNA) and of protein in Escherichia coli during glucose-lactose diauxie lag have been examined. The rate of RNA synthesis is about 7%, of the corresponding rate during exponential growth and the rate of protein synthesis 10 to 15%. Inhibition of RNA synthesis occurs to the same extent in both rel and rel(+) strains. The RNA which accumulates during 20 min in diauxie lag is composed of about 50% ribosomal and transfer RNA species and about 50% of a fraction which resembles messenger RNA (mRNA) in its heterogeneous sedimentation properties. Decay of the heterogeneous fraction occurs in the presence of glucose and actinomycin D with a half-life of 3 min, the same as that of pulse-labeled mRNA; however, during the diauxie lag, the half-life of this RNA is about 25 min. Accumulation of the heterogeneous RNA is further increased when protein synthesis is blocked by chloramphenicol. The data suggest that the disproportionate accumulation of mRNA during diauxie lag and energy source shift-down may be attributed at least in part to increased stability of mRNA, but do not rule out a preferential synthesis of mRNA.     

Fluctuations of DNA-dependent RNA polymerase and synthesis of macromolecules during the growth cycle of Novikoff rat hepatoma cells in suspension culture

The transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA-dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re-formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9–13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells. Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.     

Proceedings: Factors affecting repair of sublethal injury in frozen or freeze-dried bacteria

Conditions facilitating recovery of sublethally injured cells present in frozen or freeze-dried preparations of bacteria were investigated. Some cells of Streptococcus faecalis injured by freezing required RNA resynthesis for recovery, whereas the rest did not. Addition of 6% NaCl to control recovery medium was found to inhibit RNA synthesis completely in frozen-injured cells of S. faecalis, but not in intact ones. This seems to be a principal reason why 6% NaCl inhibits the growth of the injured cells.The recovery of viable cells of freeze-dried and stored Streptococcus thermophilus was greately increased by the addition of cysteine to the plating medium. The favorable effect of cystein was mainly due to its reducing activity. On the contrary, the addition to the plating medium of certain types of peptone, such as lactalbumin hydrolyzate, resulted in a considerable decrease of viability in frozen preparations of Vibrio metschnikovii.Thus, at present, it seems too early to try to discuss specifically the optimal composition of plating media to give the highest viability from frozen or freeze-dried preparations of bacteria.     

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. CONCLUSIONS: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

RNA Synthesis in Yersinia pestis During Growth Restriction in Calcium-Deficient Medium

Yersinia pestis requires 2.5 mM Ca(2+) for growth at 37 degrees C but not at 26 degrees C. After a shift from 26 to 37 degrees C in a Ca(2+)-deficient medium, an ordered series of metabolic alterations occur which result in transition from a growing cell to a viable but non-proliferating cell. The earliest known alteration in normal metabolism associated with this transition is a termination of net RNA synthesis. Competitive RNA/DNA hybridizations with uniformly labeled RNA and stable RNA competitor indicated identical mRNA to stable RNA ratios in growing cells and non-proliferating Ca(2+)-deprived cells. Similar hybridizations with pulse-labeled RNA demonstrated that growing cells synthesized 57% mRNA, 37% rRNA, and 5% tRNA, whereas Ca(2+)-deprived cells synthesized 95% mRNA, 4.7% rRNA, and 0.7% tRNA. After addition of radioactive uracil and rifampin to growing and Ca(2+)-deprived cells, decay of approximately 40 and 90% of the newly synthesized RNA was found for growing and Ca(2+)-deprived cells, respectively. The half-life of the mRNA was found to be 1.5 min for growing cells and 4.5 min for Ca(2+)-deprived cells. Y. pestis elicited increases in the levels of guanosine tetraphosphate and guanosine pentaphosphate in response to amino acid deprivation and yielded transient increases in the levels of these phosphorylated nucleotides after a shift from 26 to 37 degrees C. These increases were independent of Ca(2+) availability and preceded the alteration in RNA synthesis by more than 1 h. The levels of these phosphorylated nucleotides then stabilized at about 80 and 40 pmol for Ca(2+)-deprived and Ca(2+)-supplemented cultures, respectively, and did not increase further in the Ca(2+)-deprived culture at the time corresponding to the reduction in stable RNA synthesis. These findings indicate that the early lesion in RNA synthesis associated with the growth restriction of Ca(2+)-deprived Y. pestis reflects a block in stable RNA synthesis and that this effect is not mediated by guanosine tetraphosphate or guanosine pentaphosphate.     

Molecular events during the release of delta-aminolevulinate dehydratase from catabolite repression.

Transfer of exponential-phase cells of Saccharomyces cerevisiae, previously grown in 2% glucose, to a derepression medium resulted in a prompt increase in the level of delta-aminolevulinate dehydratase, the rate-limiting enzyme of heme biosynthesis under these conditions. This derepression exhibited a lag of 35 min at 23 degrees C and required the participation of both RNA and protein syntheses. Dissection of the molecular events during this lag period disclosed that RNA synthesis, rnal gene function (messenger RNA transport from nucleus to cytosol), and initiation of protein synthesis were completed within less than 10, 18, and 24 min, respectively. The potential regulation of derepression by mitochondrial gene products and mitochondrial function was probed by means of a series of isogenic, respiration-deficient (rho-, pet-, and mit-) mutants; no such regulation was found.