Photoheterotrophic utilization of acetate by the wild type and an acetate-adapted mutant of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata strain St. Louis can grow anaerobically in the light-with acetate as the carbon source. The organism is sensitive to acetate, however, initial concentrations exceeding 25 mM resulting in an extensive growth lag. Bicarbonate is not required for growth of this strain on acetate, but addition of bicarbonate shortens the lag phase in media with high initial acetate concentration. A spontaneous mutant which exhibited a minimal lag phase and rapid growth rates on acetate media was derived from strain St. Louis. This mutant possessed elevated levels of the glyoxylate cycle enzyme, isocitrate lyase.     

Accumulation of viruslike particles in a yeast mutant lacking a mitochondrial pore protein.

The lack of mitochondrial porin is not lethal in Saccharomyces cerevisiae, but it impairs some respiratory functions and, therefore, growth on nonfermentable carbon sources such as glycerol. However, after a lag phase porinless mutant cells adapt to growth on glycerol, accumulating large amounts of an 86-kilodalton (kDa) protein (M. Dihanich, K. Suda, and G. Schatz, EMBO J. 6:723-728, 1987) and of a 5-kilobase RNA. Immunogold labeling localized the 86 kDa-protein exclusively to the cytosol fraction, although most of it cosedimented with the microsome fraction in earlier cell fractionations. This discrepancy was resolved when the 86-kDa protein was identified as the major coat protein in viruslike particles (VLPs) which is encoded by a double-stranded RNA (L-A RNA). Elimination of VLPs in the original porinless strain by introduction of the mak10 or the mak3 mutation increased the respiratory defect and prolonged its lag phase on nonfermentable carbon sources. The fact that the simultaneous loss of VLPs and respiratory functions are the introduction of mak10 or mak3 occurred even in some porin-containing wild-type strains suggests that there is a link between VLP and mitochondrial functions.     

Single-Cell Dynamics Reveals Sustained Growth during Diauxic Shifts

Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.     

Physiological conditions for nitrogen fixation in a unicellular marine cyanobacterium, Synechococcus sp. strain SF1.

A marine, unicellular, nitrogen-fixing cyanobacterium was isolated from the blades of a brown alga, Sargassum fluitans. This unicellular cyanobacterium, identified as Synechococcus sp. strain SF1, is capable of photoautotrophic growth with bicarbonate as the sole carbon source and dinitrogen as the sole nitrogen source. Among the organic carbon compounds tested, glucose and sucrose supported growth. Of the nitrogen compounds tested, with bicarbonate serving as the carbon source, both ammonia and nitrate produced the highest growth rates. Most amino acids failed to support growth when present as sole sources of nitrogen. Nitrogenase activity in Synechococcus sp. strain SF1 was induced after depletion of ammonia from the medium. This activity required the photosynthetic utilization of bicarbonate, but pyruvate and hydrogen gas were also effective sources of reductant for nitrogenase activity. Glucose, fructose, and sucrose also supported nitrogenase activity but to a lesser extent. Optimum light intensity for nitrogenase activity was found to be 70 microE/m2 per s, while the optimum oxygen concentration in the gas phase for nitrogenase activity was about 1%. A hydrogenase activity was coinduced with nitrogenase activity. It is proposed that this light- and oxygen-insensitive hydrogenase functions in recycling the hydrogen produced by nitrogenase under microaerobic conditions.     

Bicarbonate Requirement for Elimination of the Lag Period of Hydrogenomonas eutropha

Carbon dioxide and oxygen concentrations have a profound effect on the lag period of chemoautotrophically grown Hydrogenomonas eutropha. Minimum lag periods and high growth rates were obtained in shaken flask cultures with a prepared gas mixture containing 70% H(2), 20% O(2), and 10% CO(2). However, excessively long lag periods resulted when the same gas mixture was sparged through the culture. The lag period was shortened in sparged cultures by decreasing both the pO(2) and the pCO(2), indicating that gas medium equilibration had not occurred in shaken cultures. The lag period was completely eliminated at certain concentrations of O(2) and CO(2). The optimum pO(2) was 0.05 atm, but the optimum pCO(2) varied according to the pH of the medium and physiological age of the inoculum. At pH 6.4, the pCO(2) required to obtain immediate growth of exponential, postexponential, and stationary phase inocula at equal specific rates was 0.02, 0.05, and 0.16 atm, respectively. With each 0.3-unit increase in the pH of the medium, a 50% decrease in the CO(2) concentration was needed to permit growth to occur at the same rate. The pCO(2) changes required to compensate for the pH changes of the medium had the net effect of maintaining a constant bicarbonate ion concentration. Initial growth of H. eutropha was therefore indirectly related to pCO(2) and directly dependent upon a constant bicarbonate ion concentration.     

Kinetic model for membrane transport. 2. Time lag and overshoot

A lag time during the period of variation in solute concentration in the receiver phase and overshoot in that in the membrane phase have been predicted to occur with a kinetic model for membrane transport which takes into account both the membrane volume and the partitioning kinetics (Makino et al., Biophys. Chem. 35 (1990) 85). The duration of the lag time becomes longest when the donor and receiver phases have the same volume. This maximum grows in length with increase in the partition coefficient, tending to be proportional to the volume fraction of the receiver phase. Moreover, it displays an increase in length with decreasing membrane volume fraction. Overshoot occurs only when the volume fraction of the receiver phase is greater than that of the donor. Overshoot is observed during the earlier stages of membrane transport when the partition coefficient is smaller or the volume fraction of the receiver phase is larger.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Resistance of Streptomyces cinnamonensis to butyrate and isobutyrate: production and properties of a new anti-isobutyrate (AIB) factor.

Butyrate and isobutyrate (after isomerization to n-butyrate) are specific precursors for the biosynthesis of monensin A in Streptomyces cinnamonensis. High concentrations of both butyrate and isobutyrate (greater than 20 and 10 mM, respectively) were toxic to S. cinnamonensis plated on solid medium. Spontaneous mutants resistant to these substances were isolated. These new strains produced monensins at even higher concentrations of butyrate or isobutyrate, with an increased yield of monensin A. S. cinnamonensis produced an anti-isobutyrate (AIB) factor, which was originally found to be excreted by some isobutyrate-resistant stains growing on solid medium containing isobutyrate. On plates, the AIB factor efficiently counteracted toxic concentrations not only of isobutyrate, but also of acetate, propionate, butyrate, 2-methylbutyrate, valerate and isovalerate against S. cinnamonensis as well as other Streptomyces species. Although the AIB factor enabled normal growth, sporulation and monensin production on plates, it did not have positive effects on submerged cultures of S. cinnamonensis with isobutyrate. The partial purification of the AIB factor was achieved. The role of the AIB factor during spore germination on solid medium containing isobutyrate or its homologues is discussed.     

Effects of Bicarbonate on Growth of Neisseria gonorrhoeae: Replacement of Gaseous CO2 Atmosphere

The effect of NaHCO₃ on the growth of Neisseria gonorrhoeae cultures was studied in a liquid and a semisolid growth medium. With a broth culture, NaHCO₃ (0.009 M) greatly reduced the lag phase and also increased the total growth. The same concentration of bicarbonate supported rapid growth when added to the semisolid medium if the plates were individually incubated in sealed plastic bags. In a container with a large air space, a higher concentration of NaHCO₃ was necessary to support growth. The assimilation of ¹⁴C-labeled NaHo₃ by growing cultures was also investigated.     

Behavior in two-phase aqueous polymer systems of L5178Y mouse leukemic cells : II. The lag and exponential phases of growth

The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran — polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude for the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.     

Bioconversion of propionic,valeric, and 4-hydroxybutyric acids into the corresponding alcohols byClostridium acetobutylicum NRRL 527

The addition of biogenic (acetate and butyrate) or abiogenic (propionate, isobutyrate, and valerate) volatile fatty acids to theClostridium acetobutylicum growth medium reduced the lag phase. Propionate and valerate were reduced to the corresponding alcohols (l-propanol andl-pentanol). Dicarboxylic acids were not metabolized, but reduced glucose utilization and solvent synthesis. A hydroxyacid, 4-hydroxybutyric acid, was quantitatively converted to 1,4-butanediol.     

The electrochemical proton potential of Bacillus alcalophilus.

Bacillus alcalophilus strain ATCC 27647 showed usual growth characteristics, when inoculated at pH 10.4. The cells entered the logarithmic phase at pH 10.3, and as growth continued, the pH dropped further to a value of 8.8 in the stationary phase. B. alcalophilus strain DSM 485 showed comparable growth only in the initial phase after the addition to fresh medium. The small initial growth period was succeeded by a long lag phase, where the pH continuously dropped. The cells resumed growth after the pH was about 10.0 and continued to grow accompanied by a further decrease of external pH. The bioenergetic parameters measured in the initial growth phase of the two strains at high pH (10.1-10.3) were nearly the same, i.e. delta pH = +97 to +110 mV, delta psi = -206 to -213 mV and delta microH+ = -109 to -103 mV. The inverted proton gradient of about 1.7-1.9 at high pH decreased, as the external pH dropped during growth. This led to an increase of the proton motive force (delta microH+), although the membrane potential (delta psi) also declined. The ATP/ADP ratio of strain DSM 485 was high (4.5-5.5) at fast growth during the initial and second growth period. The ratio declined to about 1.5 at the end of the lag phase. At the initial growth phase and at the end of the lag phase, the delta microH+ was, however, the same (approximately -106 mV) and considerably lower than in the middle of the second growth period (approximately -140 mV). Fast growth, therefore, correlates with a high ATP/ADP ratio but not necessarily with a high delta microH+. Addition of gramicidin or carbonylcyanide m-chlorophenylhydrazone stopped growth of B. alcalophilus strain DSM 485 at pH 10.3 or 9.5 and gramicidin immediately decreased the internal ATP/ADP ratio from 4.5 to 1.2 at pH 10.3.     

Comparison of biooxidation with carbon dioxide assimilation during bacterial growth on ferrous ion or elemental sulfur.

Biomass and oxygen uptake activity profiles of a mixed bioleaching culture were studied and compared at various temperatures. Bacteria were grown on ferrous ion or elemental sulfur in a Micro-Oxymax respirometer apparatus that allowed measurement of both oxygen consumption and carbon dioxide assimilation. Balanced growth was observed between 10 degrees C and 35 degrees C, with an optimum at 30 degrees C, on both energy sources. No significant growth was observed at the lowest temperature used, 5 degrees C, or at the highest temperature used, 40 degrees C. The oxygen to carbon dioxide molar yield was 50:1 when growing on ferrous ion but only 17:1 when growing on elemental sulfur. Upon transfer from a sulfide ore to a new energy source, greater numbers in the inoculum reduced the duration of the lag phase. Lag phase duration was also reduced by proximity to the optimum growth temperature. A longer lag phase decreased the achievable growth rate of the cells exponentially, significantly affecting biooxidation activity.     

Effect of culture conditions on batch growth ofPseudomonas fluorescens on olive oil

Summary Batch cultures ofPseudomonas fluores-cens were grown in minimal medium with olive oil as the sole carbon source. When olive oil me-dium was inoculated with cells from nutrient broth there was an initial lag phase followed by logarithmic growth. The duration of the lag phase was influenced by the incubation temperature and the growth phase of the inoculum. Both factors are known to affect lipase induction during growth in fat-free media. Maintenance of condi-tions reported to be conducive to lipase produc-tion in cultures used for inoculation ensured a minimal lag before logarithmic growth com-menced on olive oil. Growth on oil occurred when the culture was maintained at pH 6 or 7, but did not occur at pH 5 or 8.     

Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.     

Pyruvate-dependent diauxic growth of Rhodospirillum rubrum in light.

When Rhodospirillum rubrum mutant C was first exposed to radiant energy after long-term anaerobic dark growth, the cells often exhibited a diauxic growth response. This happened with pyruvate in the medium and when cultures were exposed to a less-than-growth-saturating white light intensity of about 6,460 lx. Under the growth-saturating light condition, mutant C photometabolized and growth was not affected by Na hypophosphite, an inhibitor of pyruvate fermentation. In lower intensity light, in which diauxie occurred, initial (phase I) growth occurred by fermentation of Na pyruvate and was sensitive to Na hypophosphite inhibition. Once pyruvate was depleted, phase I growth stopped, the bacteriochlorophyll content of the cells began to increase from about 3 nmol/mg of protein, and growth finally resumed phototrophically (phase II). The lag period and phase II growth were influenced by radiant energy. By changing the white light intensity from 2,150 to 753 lx between experiments, the duration of both the lag period and the generation time of cells in phase II growth increased. Diauxic growth was pyruvate dependent. It occurred with pyruvate even if malate, a photometabolizable substrate, was added to the growth medium. Moreover, the biphasic growth response was reversible. It was observed not only with R. rubrum mutant C grown cells photosynthetically, but also when other strains of R. rubrum were placed in pyruvate medium under lowered light conditions. Only R. rubrum S1 did not exhibit the typical pyruvate-dependent diauxic growth response.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

Individual-based modelling of bacterial cultures to study the microscopic causes of the lag phase

The lag phase has been widely studied for years in an effort to contribute to the improvement of food safety. Many analytical models have been built and tested by several authors. The use of Individual-based Modelling (IbM) allows us to probe deeper into the behaviour of individual cells; it is a bridge between theories and experiments when needed. INDividual DIScrete SIMulation (INDISIM) has been developed and coded by our group as an IbM simulator and used to study bacterial growth, including the microscopic causes of the lag phase. First of all, the evolution of cellular masses, specifically the mean mass and biomass distribution, is shown to be a determining factor in the beginning of the exponential phase. Secondly, whenever there is a need for an enzyme synthesis, its rate has a direct effect on the lag duration. The variability of the lag phase with different factors is also studied. The known decrease of the lag phase with an increase in the temperature is also observed in the simulations. An initial study of the relationship between individual and collective lag phases is presented, as a complement to the studies already published. One important result is the variability of the individual lag times and generation times. It has also been found that the mean of the individual lags is greater than the population lag. This is the first in a series of studies of the lag phase that we are carrying out. Therefore, the present work addresses a generic system by making a simple set of assumptions.     

NMR spin-echo study of the lyophilized biomass of Candida utilis yeasts at different growth stages]

Characteristics of the mobile water fraction isolated from some intracellular structures of freeze-dried cells of Candida utilis were studied as a function of the growth phase by the NMR spin-echo method. Part of the mobile water fraction in the log phase decreases down to ten percent of the biomass weight as compared with 2,5-4% in the stationary phase and in the beginning of the lag phases. Mobility of this water is invariable. A considerable decrease of the mobile water fraction is observed in the post-stationary phase too. A decrease of the mobile water fraction correlates with a decrease of the durability of C. utilis cells and is connected probably with some defects in the membrane structure of the cells and their incomplete development in the log phase of the growth.