Self-reproduction and doubling time limits of different cellular subsystems.

Ribosomes which can self-replicate themselves practically autonomously in beneficial physicochemical conditions have been recognized as the central organelles of cellular self-reproduction processes. The challenge of cell design is to understand and describe the rates and mechanisms of self-reproduction processes of cells as of coordinated functioning of ribosomes and the enzymatic networks of different functional complexity that support those ribosomes. We show that doubling times of proto-cells (ranging from simplest replicators up to those reaching the size of E. coli) increase rather with the number of different cell component species than with the total numbers of cell components. However, certain differences were observed between cell components in increasing the doubling times depending on the types of relationships between those cell components and ribosomes. Theoretical limits of doubling times of the self-reproducing proto-cells determined by the molecular parameters of cell components and cell processes were in the range between 6-40 min.

Single-cell model of prokaryotic cell cycle.

One of the recognized prokaryotic cell cycle theories is Cooper-Helmstetter (CH) theory which relates start of DNA replication to particular (initiation) cell mass, cell growth and division. Different aspects of this theory have been extensively studied in the past. In the present study CH theory was applied at single cell level. Universal equations were derived for different cell parameters (cell mass and volume, surface area, DNA amount and content) depending on constructivist cell cycle parameters (unit mass, replication and division times, cell age, cell cycle duration) based on selected growth laws of cell mass (linear, exponential). The equations derived can be integrated into single-cell models for the analysis and design of bacterial cells.

Specific growth rate dependent transcriptome profiling of Escherichia coli K12 MG1655 in accelerostat cultures.

Specific growth rate dependent gene expression changes of Escherichia coli K12 MG1655 were studied by microarray and real-time PCR analyses. The bacteria were cultivated on glucose limited minimal medium using the accelerostat method (A-stat) where starting from steady state conditions (chemostat culture) dilution rate is constantly increased. At specific growth rate (mu) 0.47h(-1), E. coli had focused its metabolism to glucose utilization by down-regulation of alternative substrate transporters expression compared to mu=0.3h(-1). It was found that acetic acid accumulation began at mu=0.34+/-0.01h(-1) and two acetate synthesis pathways - phosphotransacetylase-acetate kinase (pta-ackA) and pyruvate oxidase (poxB) - contributed to the synthesis at the beginning of overflow metabolism, i.e. onset of acetate excretion. On the other hand, poxB, pta and ackA expression patterns suggest that pyruvate oxidase may be the only enzyme synthesizing acetate at mu=0.47h(-1). Loss of glucose and acetate co-utilization represented by down-regulation of acs-yjcH-actP operon between specific growth rates 0.3-0.42h(-1) and acetic acid accumulation from mu=0.34+/-0.01h(-1) allows one to surmise that the acetate utilization operon expression might play an important role in overflow metabolism.

Steady state growth space study of Lactococcus lactis in D-stat cultures.

Growth space of Lactococcus lactis subsp. lactis IL1403 was studied at constant growth rate using D-stat cultivation technique. Starting from steady state conditions in a chemostat culture (mu = 0.2 h(-1)), the pH and/or temperature were continuously changed in the range of 5.4-6.4 and 26-34 degrees C, respectively, followed by the return to the initial environmental conditions. Based on substrate consumption and product formation yields and expression changes of 1,920 genes, it was shown that changes of physiological state were not dependent on the direction of movement (from pH 6.3 to 5.4 or from 5.4 to 6.3), showing that quasi steady state values in D-stat corresponded to the steady state values in chemostats. Relative standard deviation of growth characteristics in triplicate D-stat experiments was below 10%. Continuing the experiment and reestablishing initial growth conditions revealed in average 7% difference (hysteresis) in growth characteristics when comparing chemostat steady state cultures prior and after the change of environmental conditions. Similarly, shifts were also seen at gene expression levels. The large amount of quantitatively reliable data obtained in this study provided a new insight into dynamic properties of bacterial physiology, and can be used for describing the growth space of microorganisms by modeling cell metabolism.

Quasi steady state growth of Lactococcus lactis in glucose-limited acceleration stat (A-stat) cultures.

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h(-2) after initial stabilization of the cultures in chemostat at D = 0.2-0.3 h(-1). It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used-at an acceleration rate 0.003 h(-2) the quasi steady state growth was observed until mu (crit) = 0.59 h(-1), which is also the mu (max) value for the culture. Lower values of mu (crit) were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h(-1) was immediately disrupted after initiating acceleration at the highest acceleration rate studied-0.06 h(-2). Observation was made that differences [Delta(mu - D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h(-2) (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate-it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S (0) = 5 g L(-1) glucose instead of S (0) = 10 g L(-1). It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility-Delta(mu - D) was less than 5%, standard deviations of biomass yields per ATP produced (Y (ATP)), and biomass yields per glucose consumed (Y (XS)) were less than 15%.

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study.

The effect of individual environmental conditions (pH, pO(2), temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol(-1)) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y(ATP) and Q(ATP) values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO2) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y(ATP)) and specific growth rate, however the specific ATP production rate (Q(ATP)) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l(-1)), propanol (>2 g l(-1)) or NaCl (>15 g l(-1)) lead at first to the decrease in the specific growth rate and Q(ATP), while Y(ATP) was affected only at higher concentrations. The observed decrease in Q(ATP) was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h(-1) and Y(ATP) from 12.5 to 18.5 mol ATP g dwt(-1). This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells.

The advantage of using 13C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that 13C-[2] acetate or 13C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using 13C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/13C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.