Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase-coupled transition of E. coli phenotype is discontinuous.     

Enhanced fitness of recombinant protein synthesis in the stationary phase of Escherichia coli batch cultures

The ability to synthesize both recombinant and homologous cell proteins has been studied during the stationary phase of E. coli batch cultures. The anabolic potential of the culture dramatically decreases when entering into the stationary phase but slightly recovers several hours latter. In addition, CI857-controlled production of -galactosidase is transiently enhanced at late stages of the stationary phase. These results show a non-synchronous distribution of the biosynthetic resources throughout culture growth phases, favouring the production of recombinant proteins in both exponentially growing and aged, stationary cells.     

Oxidative side reactions during dansylation of SH-compounds.

Dansyl chloride can act as an oxidizing agent on compounds which are easily oxidized. During the reaction of mercaptanes, e.g. cysteine, homocysteine, cysteamine, with dansyl chloride, the corresponding disulfides are formed and dansyl chloride is reduced to 5-dimethylaminonaphthalene 1-sulfinic acid. This reaction is so rapid that the normal dansylation can take place only after complete oxidation of all SH-compounds. Therefore only the dansyl derivatives of the corresponding disulfides are formed during normal dansylation of SH-compounds. If different SH-compounds are present in the reaction mixture mixed disulfides are formed as well. These can be separated by microchromatography on 3 X 3 cm micropolyamide sheets. Dependent on the concentration of dansyl chloride, 6 or even 15 different dansylated disulfides are formed from three different SH-compounds so that interpretation of these chromatograms is difficult. The actual dansylmercaptanes (e.g., dansylcysteine, dansylhomocysteine, dansylcysteamine) can be prepared by reduction of the dansylated disulfides with suitable reducing agents.     

Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection

Low-molecular-mass thiols, such as glutathione (GSH), and their associated disulfides are ubiquitous in nature, and based upon the many known functions of these compounds, their identification and accurate measurement is essential. Our objectives were to develop a simple method for the simultaneous measurement of thiols and disulfides in biological samples using HPLC with dual electrochemical detection (HPLC-DED). Particular emphasis was placed on the applicability to a wide variety of important GSH-related thiols and disulfides, including γ-Glu-Cys, Cys-Gly, their disulfides, and the mixed disulfide of glutathione and cysteine (CSSG), validation on different types of biological samples, maintenance of chromatographic resolution and reproducibility with routine and extended use, and enhancement of assay sensitivity. To this end, optimal HPLC conditions including mobile phase, column, and electrode polishing procedures were established and the method was applied to, and validated on a variety of biological samples. This improved methodology should prove to be a useful tool in studies on the metabolism of GSH and other thiols and disulfides and their role in cellular homeostasis and disease processes.     

Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems

A series of simple methodologies for the determination of the redox status of low molecular weight and protein thiols in biological systems is described. Based centrally upon the use of monobromobimane, we describe a standard in situ derivatisation procedure simultaneously resulting in maximal recovery of both free, reduced low molecular weight and bromobimane accessible protein thiols as their corresponding bimane adducts from intact biological systems. Test systems include isolated and cultured cells, tissue homogenates and body fluids such as blood plasma. Quantitation of the bimane adducts of cysteine and glutathione is achieved by reversed phase high performance liquid chromatography, whereas quantitation of the corresponding adducts of protein thiols is achieved by fluorescence spectroscopy following protein precipitation. Full validation data for quantitative estimates are described. Additionally we have coupled these procedures to prederivatization denaturation treatments of biological protein samples in order to quantitate pools of protein thiols which are inaccessible to bromobimane in samples of native protein. We have also coupled these procedures with prederivatization reductions of biological systems under study with dithiothreitol, rendering simultaneously both oxidized low molecular weight thiols and oxidized protein thiols accessible to derivatisation with monobromobimane. Thus, we have obtained quantitative determinations of cysteine and glutathione present in mixed disulfides with protein and in soluble low molecular weight disulfides and estimates of intraprotein disulfides in a number of test biological systems.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Adenylate energy charge in Saccharomyces cerevisiae during starvation.

Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.     

Colony dimorphism associated with stress resistance in Oenococcus oeni VP01 cells during stationary growth phase

The resistance to stresses as starvation, the presence of ethanol, sulfite and low pH, is a fundamental prerequisite for starter cultures used to induce malolactic fermentation in wine. In order to evaluate stress resistance of cells undergone starvation, cells viability in laboratory cultures of Oenococcus oeni VP01 strain was monitored during prolonged stationary growth phase. Once entered the stationary phase, strain VP01 showed 99% reduction of cell viability within 4 days. The remaining cells population maintained viability over 70 days and, when plated on agar medium, generated small colonies. The occurrence of this phenomenon was associated to stress resistance, since 10-day-old cells resulted more resistant than 3-day-old cells to ethanol and low pH conditions. No genomic mutations were revealed by pulse-field gel electrophoresis (PFGE) analysis in aged cultures. Total protein analysis by bidimensional electrophoresis highlighted differential protein expression in cultures differentially aged. It was demonstrated that O. oeni starving cultures at the stationary phase are constituted by dynamic cell populations. These results offer interesting perspective for a better understanding of cells behavior when inoculated in wine.     

Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Growth-phase regulation of the Escherichia coli thioredoxin gene

The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170. Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures. The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp). RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression. Increased expression of relA, which increases ppGpp concentration, increases trxA expression.     

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.     

Survival of Streptococcus pyogenes under stress and starvation

The ability of Streptococcus pyogenes to enter a quiescent state, similar to the stationary phase of lab cultures, is believed to be an important factor in its ability to persist within the host and to subsequently cause disease. Using a model broth system, we determined that after entering the stationary phase, there was a 99.99% reduction in cell viability over a 4-day period, following which the cells appeared to enter a resistant starvation state where cell numbers remained constant over the subsequent 3-4 weeks. This starvation response was induced by carbon or phosphorous limitation, but not by nitrogen limitation in the form of amino acids where cells became non-culturable after 4 days. Amino acid utilization in the absence of a carbon source may be an essential factor for the long-term survival of this bacterium in the stationary phase. Early stationary phase cells showed a greater resistance to oxidative and pH stress compared to 24-h-starved cultures. There was evidence for the formation of a viable but non-culturable state as indicated by a comparison of the numbers of cells with a functional membrane potential (rhodamine 123) against culturable cells on either Todd Hewitt broth agar or sheep blood agar. Long-term survival of S. pyogenes was dependent on both cell wall and protein synthesis, suggesting that starving cultures are a dynamic cell population.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

Recovery from nutrient starvation by a marine Vibrio sp.

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

Recovery from nutrient starvation by a marine Vibrio sp

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

Changes of ploidy during the Azotobacter vinelandii growth cycle.

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory.