Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein.

The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Analysis of the physiological control of replication of ColE1-type plasmids

The physiology of ColE1-type plasmid replication in a growing host has been examined both theoretically, using computer simulation, and experimentally, by observing replication of the plasmid pBR322 after a nutritional shift-up from glycerol minimal medium (doubling time 71 min) to LB medium (doubling time 24 min). The theory was based on a negative control model and uses three rate equations: for the accumulation of cell mass, for the accumulation of the replication inhibitor, and for the rate of plasmid synthesis. The implications of the theory were explored by simulating the effects of changes in the expression of replication control genes. The nutritional shift-up experiment showed that plasmid replication was blocked immediately after the shift for about half a mass doubling time; after that time, replication rapidly increased until plasmid numbers per unit volume of culture parallelled the increase in culture mass. After the establishment of steady-state growth in the post-shift medium, the plasmid concentration (plasmids per cell mass) was reduced in comparison to pre-shift growth in the same proportion as the culture doubling time. The results showed that plasmid replication factors are under metabolic control and that the changes in the control of these factors compensate one another during steady-state growth, but not immediately after the medium shift.     

The regulation of RNA synthesis in yeast II: Amino acids shift-up experiments

Summary A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15–20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of the regulation of RNA synthesis are discussed.Paper I in this series is Oliver and McLaughlin (1977)     

Growth and cell division of Mycobacterium avium

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.     

The inefficiency of ribosomes functioning in Escherichia coli growing at moderate rates

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.     

Regulation of RNA synthesis in Neurospora crassa : An analysis of a shift-up

A shift-up transition of growth from acetate to glucose is analyzed in Neurospora crassa. The rates of DNA and of protein accumulations remain at the preshift values for about 2 h, afterwards they increase to the rate characteristic of the new medium. The rate of RNA accumulation increases markedly 30 min after glucose addition initially at a rate greater than that of the new exponential growth which is achieved later on. An increase of the level of ribosomal proteins accompanies the increase of the rRNA content of the shifting cells, and 2–2.5 h after the shift the ribosomal level has reached the value characteristic of the new steady state of growth. The rate of rRNA methylation, which is strictly proportional to rRNA synthesis, remains almost unchanged in the 30 min following the shift; thereafter it increases to values greater than the final rate. It is interesting that the rate of rRNA synthesis is enhanced above the value typical of the new steady state as long as the ribosome level in the cells is below that characteristic of the new steady state, as if a compensatory mechanism were active.     

Mammal cells double their total RNAs against diabetes, ischemia reperfusion and malaria-induced oxidative stress

Total cellular RNA level is stable usually, although it may increase gradually during growth or decrease gradually under certain stressors. However, we found that mammal cell RNAs could be doubled within 24 h in response to free heme accumulation (ischemia reperfusion and malaria infection) or a high level of glucose treatment (diabetes). Clinical investigations in rats showed that pretreatment with heme (24 h for doubling total RNAs) alleviated oxidative damages caused by diabetes, and pretreatment with glucose (24 h for trebling total RNAs) alleviated oxidative damages caused by ischemia reperfusion or malaria infection. Therefore, this rapid RNA amplification may play an important role in mammal adaptation to diabetes, ischemia reperfusion and malaria infection-derived oxidative stress. This rapid RNA amplification is derived from glucose and heme, but not from their accompanying reactive oxygen species. Hexokinases endure glucose-derived reactive oxygen species accumulation but are not related glucose-derived RNA amplification. In contrast, the TATA box-binding protein (TBP) mediates all glucose- and heme-induced RNA amplification in mammal cells.     

Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens.

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.     

Effect of Medium Composition on the Denitrification of Nitrate by Paracoccus denitrificans

The course of denitrification of nitrate in static cultures of Paracoccus denitrificans was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by P. denitrificans is not stable and depends on the nutritional composition of the culture medium.     

Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth.

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.     

Cellular and morphological changes at the terminal shoot apex of the short-day plant Pharbitis nil during the transition to flowering

Changes in morphology and measurements of cell doubling time were recorded for the first time in the terminal shoot apex of the short-day plant, Pharbitis nil Chois. ( Ipomea nil L.) cv. Violet, undergoing the floral transition. A treatment comprising 48 h darkness given to 4-day-old plants resulted in 100% flowering at the shoot terminal meristem. An inhibitory treatment comprising two 5 min red night-breaks during the 48 h dark period was used to discriminate between events essential for flowering, and those changes resulting from shifts from light to darkness and vice versa. Morphology was studied using both light microscopy and scanning electron microscopy. Cell doubling times were measured using the colchicine accumulation of metaphases method. An increase in the rate of primordial initiation, a change in the divergence angle and a change in phyllotaxis occurred during the floral transition. Moreover, the apex widened and flattened following the inductive dark treatment; the cell doubling time decreased in the peripheral zone and increased in the central zone of these pre-floral meristems.     

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.     

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.     

Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism

An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D = 0.03 h(-1)) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D = 0.01 h(-1)) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.     

Isolation and characterization of a novel sphingomonad capable of growth with chrysene as sole carbon and energy source

A bacterial strain able to grow in pure culture with chrysene as sole added carbon and energy source was isolated from PAH-contaminated soil after successive enrichment cultures in a biphasic growth medium. Initially, growth occurred in the form of a biofilm at the interface between the aqueous and non-aqueous liquid phases. However, after a certain time, a transition occurred in the enrichment cultures, with growth occurring in suspension and a concomitant increase in the rate of chrysene degradation. The strain responsible for chrysene degradation in these cultures, named Sphingomonas sp. CHY-1, was identified by 16S rDNA sequencing as a novel sphingomonad, the closest relative in the databases being Sphingomonas xenophaga BN6T (96% sequence identity). Both these strains clustered with members of the genera Sphingobium and Rhizomonas, but could not be categorically assigned to either genus. Sphingomonas sp. CHY-1 was characterized in terms of its growth on chrysene and other PAH, and the kinetics of chrysene degradation and 14C-chrysene mineralization were measured. At an initial chrysene concentration of 0.5 g l(-1) silicone oil, and an organic/aqueous phase ratio of 1:4, chrysene was 50% degraded after 5 days incubation and 97.5% degraded after 35 days. The protein content of cultures reached a maximum value of 11.5 microg ml(-1) aqueous phase, corresponding to 92 mg g(-1) chrysene. 14C-labelled chrysene was 50% mineralized after 6-8 weeks incubation, 10.7% of the radioactivity was incorporated into cell biomass and 8.4% was found in the aqueous culture supernatant. Sphingomonas sp. CHY-1 also grew on naphthalene, phenanthrene and anthracene, and naphthalene was the preferred substrate, with a doubling time of 6.9 h.     

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.