Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Dynamic proteome changes of Shigella flexneri 2a during transition from exponential growth to stationary phase

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.     

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria

Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Hydrophobicity development,alkane oxidation,and crude-oil emulsification in a Rhodococcus species

The relationship between the phenomena alkane oxidation, extreme hydrophobicity of the cell surface, and crude-oil emulsification in Rhodococcus sp. strain 094 was investigated. Compounds that induce the emulsifying ability simultaneously induced the cytochrome P450-containing alkane oxidizing system and the transition from low to high cell-surface hydrophobicity. Exposed to inducers of crude-oil emulsification, the cells developed a strong hydrophobic character during exponential growth, which was rapidly lost when entering stationary phase. The loss in hydrophobicity coincided in time with the crude-oil emulsification, indicating that the components responsible for the formation of cell-surface hydrophobicity act as excellent emulsion stabilisers only after release from the cells. Rhodococcus sp. strain 094 possessed three distinct levels of cell-surface hydrophobicity. One level of low hydrophobicity was characteristic of cells in late stationary phase and was independent of growth substrate. A second and more hydrophobic level was observed for cells in exponential phase grown on water-soluble substrates, while a third level, characterised by extreme cell hydrophobicity, was observed for cells in exponential phase cultivated on hydrophobic substrates such as hexadecane. The production of the oil-emulsifying agents seems to require external sources of nitrogen and phosphate.     

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.     

Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3'',5''-monophosphate.

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.     

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (～6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.     

Cellular control of growth in cultures of Tetrahymena. 3. The influence of Fe-medium and aeration on cellular ultrastructure.

Cultures of Tetrahymena pyriformis were grown with and without the addition of Fe and with no aeration. The same cultures were used both for determinations of the cellular Fe and Ca concentrations and the exchange of Ca (reported earlier), and for the ultrastructural study. In all cultures there was an increase in rounded, tubuli-deficient mitochondria at the transition to the prestationary growth phase. In the non-aerated culture (high cellular Fe content) these changes were less marked. In the Fe-deficient culture, however, these mitochondrial changes were seen as early as the late exponential growth phase, and tubuli-degeneration then increased during the prestationary growth phase. In this culture an irregular infolding of the outer mitochondrial membranes occurred. These changes are discussed in correlation with cytochromes, Fe-dependent desaturation of fatty acids, and high Ca concentration (non-aerated cells). During the prestationary growth phase of the non-aerated culture there was a marked increase in the amount of mitochondrial tubuli. In the organelles identified as peroxisomes there was, in all the cultures, an increased granular density of the matrix at the transition to the prestationary growth phase (in the Fe-deficient cells this occurred in the late exponential growth phase). This was correlated with an increased peroxisomal activity. The Fe-deficient culture has cells with very irregularly formed peroxisomes. This organelle was in all the cell-material very sensitive to the method of fixation. In the Fe-deficient late exponential cells there are long, bifacial pieces of RER (one side rough and the other smooth) which later undergo degradation. Many lipid droplets were seen at the ends of RER. Structures which in the literature have been called 'ergoplasm-like stacks of flattened rough cisternae' were found in the non-aerated exponential cells. They were absent from prestationary cells. In all the cultures there was an increased aggregation of the ribosomes in the cytoplasm during the prestationary growth phase. This was correlated with the accumulation of Ca in this cell fraction. An explanation is suggested regarding the earlier reported variations in the exchange of Ca, found in all the types of cultures at the transition to the prestationary growth phase.     

Physiological and biochemical characterization of a suspensionculture system for sustained exponential growth of Nicotiana silvestris

A strong approach to understanding the regulation of enzymes in metabolic pathways, such as those responsible for amino acid biosynthesis, is to follow enzyme levels throughout the growth curve of higher plant cells in suspension culture. The rise and fall of enzyme levels can be traced as a function of physiological stage of growth Subculturing, as typically carried out by low-factor dilution of stationary phase cells, yields a system suitable for the study of changes in enzyme and metabolite levels that accompany the transition from stationary-phase physiology to exponential-phase physiology. However, the short duration of exponential growth in such subculture protocols is inadequate to avoid carryover effects from previous stationary-phase physiology. Suspension cultures of Nicotiana silvestris Speg, et Comes (2N = 24) were used to demonstrate substantial carryover levels of acid phosphatase, alkaline phosphatase and protease activities. A subculture routine is described for maintaining cell populations in exponential phase indefinitely. About 10 generations of sustained exponential growth is required to approach a true balanced state of exponential growth. Such exponential phase populations consist of cells termed EE cells. EE-cell populations were similar to cells that have been in exponential phase for only a few generations (E cells), with respect to doubling time (about 40 h) and to minimal density of diluted populations able to resume growth (about 500 cells ml^?1). EE cells possess a high content of soluble protein; they are smaller and more aggregated than are E cells. Upon dilution into fresh medium, EE cells resume exponential growth without a lag. In contrast to E cells, EE cells exhibit properties of balanced growth, since proportionate increases in cell number, dry weight, wet weight and packed-cell volume were observed. E cells, sampled at different elapsed times of growth, are likely to differ in metabolite, enzyme and cell properties, whereas EE cells exhibit near-constant properties.     

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.     

Developmental changes in soluble proteins during the early development of Dictyostelium discoideum

Changes in the pattern of soluble proteins that accumulatedat the growth phase, interphase and late-aggregation phase ofthe cellular slime mold Dictyostelium discoideum were studiedby two-dimensional polyacrylamide gel electrophoresis. Amongthe 300 proteins detected during the early development, themost soluble do not change during the growth and aggregationphases, but about 90 proteins show changes in their relativeintensities on staining. During the transition from growth tothe interphase, the predominant changes were the disappearanceof 16 spots, the decrease in 30 spots, the appearance of 13new spots, and the increase in 14 spots. In contrast, from theinterphase to the late-aggregation phase, there were remarkableprogressive increases in 13 spots, an overall increase in 6spots, a decrease in 16 spots, the appearance of 8 new spotsand the disappearance of 4 spots. (Received July 13, 1979; )     

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al ., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.     

Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur

The growth kinetics of Thiobacillus thiooxidans on elemental sulfur in batch cultures at 30(deg)C and pH 1.5 was studied by measuring the time courses of the concentration of adsorbed cells on sulfur, the concentration of free cells suspended in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate ions, the surface concentration of adsorbed cells per unit mass of sulfur approached a maximum value (maximum adsorption capacity of sulfur particles) whereas the concentration of free cells continued to increase with time. There was a close relationship between the concentrations of free and adsorbed cells during the microbial sulfur oxidation, and the two cell concentrations were well correlated by the Langmuir isotherm with adsorption equilibrium constant K(infA) and maximum adsorption capacity X(infAm) of 2.10 x 10(sup-9) ml per cell and 4.57 x 10(sup10) cells per g, respectively. The total concentration of free and adsorbed cells increased in parallel with the amount of sulfate formed. The total growth on elemental sulfur gave a characteristic growth curve in which a linear-growth phase followed the period of an initial exponential phase. The batch rate data collected under a wide variety of inoculum levels (about 10(sup5) to 10(sup8) cells per ml) were consistent with a kinetic model assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration, X(infA), of adsorbed cells and the fraction, (theta)(infV), of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were estimated from the experimental data, and the specific growth rate, (mu)(infA), and growth yield, Y(infA), were 2.58 day(sup-1) and 2.05 x 10(sup11) cells per g, respectively. The proposed model and the parameter values allowed us to predict quantitatively the surface attachment of T. thiooxidans cells on elemental sulfur and the bacterial growth in both initial exponential and subsequent linear phases. The transition from exponential to linear growth was a result of two competing factors: an increase in the adsorbed-cell concentration, X(infA), permitted a decrease in the unoccupied-site fraction, (theta)(infV).     

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.     

Regulation of protein turnover versus growth state: ascites hepatoma as a model for studies both in the animal and in vitro

Cell protein turnover states as related to growth phase have been analyzed in a rat ascites hepatoma (Yoshida AH-130), which after transplantation entered a period of exponential growth, followed by a quasi-stationary state. Evaluation of AH-130 cell protein turnover in the animal (slow-turnover protein pool) was combined with rapid assays of proteolytic rates of cells transferred in vitro. Protein accumulation in the exponential phase reflected the balance between sustained synthetic rates and relatively low degradative rates. Cessation of growth resulted from convergent reduction of synthesis (from 3.10 to 1.49%/h) and enhancement of protein breakdown (from 0.61 to 1.43%/h). Endogenous proteolytic rates in vitro were very close to the above degradation rates. As shown by incubation with ammonia or other lysosomal inhibitors, the acidic vacuolar pathway for protein degradation, while totally suppressed in exponential tumor cells, was activated in cells from stationary tumors to such an extent that it fully accounted for the enhanced proteolysis. In contrast, energy metabolism inhibitors were effective on cells in either growth state, the residual ongoing proteolysis being similar in both cells. The possible contribution of cell death to activation of the acidic vacuolar proteolysis in stationary tumors is discussed.     

Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.     

Concentration and metabolism of cyclic AMP during the early stages of hepatoma 22a growth]

Intracellular levels of cyclic AMP (cAMP), adenylate cyclase, and cAMP-phosphodiesterase activities at lag-period, exponential and stationary growth phases of hepatoma 22a were determined. It was shown that the transition of tumour cells from the lag-period to the exponential phase of growth was accompanied by the two-fold decrease of intracellular cAMP level on account of drastic activation of cAMP phosphodiesterase. Subsequently the cAMP level lowered more slowly until the cells entered the stationary phase of growth. In view of the fact that the adenylate cyclase activity failed to change at different growth phases of hepatoma 22a, it seems very proballe that the rise of cAMP phosphodiesterase activity could be a signal for the exit of tumour cells from the lag-period and their entrance into the mitotic cycle.