Osmotic significance of glycerol accumulation in exponentially growing yeasts

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Induction kinetics of RNA and proteins in exponentially growing organisms.

A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing organisms is derived, and the cellular concentrations of the induced macromolecules at a given time after induction are related to three parameters: the fraction of the synthesis of these macromolecules in total synthesis, the half life of the inducible macromolecules, and the generation time of the organisms. The model predicts that the concentrations of the inducible macromolecules reach one half of the maximum induction level within one generation time after the onset of the induction. The model also predicts that induction curves of proteins are parabolic when their mRNAs are short-lived, but sigmoid when they are stable. Observed induction curves of beta-galactosidase in Escherichia coli cells fit in the theoretical induction curves.     

Iron accumulation by saccharomycete yeasts growing on media with an elevated iron content

In the absence of the artificial complexons of iron the intracellular iron content in yeast Saccharomyces cerevisiae are determined by the concentration of ions Fe(II) in medium. As iron is accumulated by yeast mainly in threevalency state the principal internal factor determining the intracellular iron content with existing concentration of Fe(II) in medium can be supposed to be redaction-oxidation state of yeast cell.     

Silver tolerance and accumulation in yeasts

Summary Debaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO₃. Although Ag⁺ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag⁺ to Ag⁰ resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO₃-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag⁰ although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag⁺ reduction to Ag⁰.     

Isoniazid resistance of exponentially growing Mycobacterium smegmatis biofilm culture

Biofilm growth of Mycobacterium smegmatis was found to be unaffected at an isoniazid concentration that inhibited growth of planktonic bacilli (i.e. at isoniazid minimum inhibitory concentration=10 microg ml(-1)). Significant growth (50% of drug-free control) of biofilms was observed at up to 40 microg ml(-1) and the MIC for biofilm growth showed an increase to up to 80 microg ml(-1) isoniazid. Thus, the biofilm growth modus appears to be a strategy for replicating bacilli to evade the onslaught of antibacterials.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Probability of resistance evolution for exponentially growing virus in the host

Chemotherapy for tumor and pathogenic virus often faces an emergence of resistant mutants, which may lead to medication failure. Here we study the risk of resistance to evolve in a virus population which grows exponentially. We assume that infected cells experience a "proliferation event" of virus at a random time and that the number of newly infected cells from an infected cell follows a Poisson distribution. Virus starts from a single infected cell and the virus infection is detected when the number of infected cells reaches a detection size. Initially virus is sensitive to a drug but later acquires resistance by mutations. We ask the probability that one or more cells infected with drug-resistant virus exist at the time of detection. We derive a formula for the probability of resistance and confirm its accuracy by direct computer simulations. The probability of resistance increases with detection size and mutation rate but decreases with the population growth rate of sensitive virus. The risk of resistance is smaller when more cells are newly infected by viral particles from a single infected cell if the viral growth rate is the same.     

Evolution of linkage disequilibrium of the founders in exponentially growing populations

Evolution of linkage disequilibrium of the founders in exponentially growing populations was studied using a time-inhomogeneous It? process model. The model is an extension of the diffusion approximation of the Wright-Fisher model. As a measure of linkage disequilibrium, the squared standard linkage deviation, which is defined by a ratio of the moments, was considered. A system of ordinary differential equations that these moments obey was obtained. This system can be solved numerically. By simulations, it was shown that the squared standard linkage deviation gives a good approximation of the expectation of the squared correlation coefficient of gamete frequencies. In addition, a perturbative solution was obtained when the growth rate is not large. By using the perturbation, an asymptotic formula for the squared standard linkage deviation after a large number of generations was obtained. According to the formula, the squared standard linkage deviation tends to be 1/(4Nc), where N is the current size of the population and c is the recombination fraction between two loci. It is dependent on neither the initial effective size, the growth rate, nor the mutation rate. In exponentially growing populations, linkage disequilibrium will be asymptotically the same as that in a constant size population, the effective size of which is the current effective size.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Lysosome changes in exponentially growing, synchronized and differentiating L-cell cultures

Using supravital fluorescent staining of lysosomes with Euchrysine 3R, the morphology of these organelles was studied in L cells examined from cultures being at different growth phases in the course of cell cycle and after adipocyte conversion of L cells due to the 60% bovine serum administration. As cells were passing from the lag-phase to the stationary phase of culture growth, the number of lysosomes was seen to increase. The appearance of large lysosomes is characteristic of cells in confluent and senescent cultures. During G1-period, lysosomes are often confined to the perinuclear area of L-cells, to be extended later during S and G2-periods. In dividing cells, these are commonly seen scattered throughout the cell periphery, around the mitotic spindle. In cells undergoing differentiation, within 4-7 days the seeding in the medium supplemented with 60% bovine serum, the number of lysosomes became augmented to be gradually reduced during the next 10-15 days, concomittantly with the accumulation of lipid drops in the cell cytoplasm. The activity of the Golgi complex and the intensity of autophagy are discussed as possible regulation points of lysosome formation during the cell growth.     

Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium discoideum

The synthesis of the lectin, discoidin I, by vegetative cells of Dictyostelium discoideum (strain NC4) was monitored using immunoblot analysis and indirect immunofluorescence. Suspension cultures were used, so that the D. discoideum cell density and the concentration of bacteria could be controlled. Discoidin-I production was found to be a function of the relative densities of D. discoideum cells and food bacteria. Synthesis was initiated in exponentially growing D. discoideum cells approximately three generations before depletion of the food supply. In the growth medium of cells producing discoidin I, a soluble activity was detected that caused low-density cells to begin discoidin-I synthesis. This activity was not dialyzable and was destroyed by heat. A similar activity was produced by AX3 cells during axenic growth. Density-dependent induction of other 'early developmental' proteins was also detected in wild-type cells. These findings suggest that the expression of several 'early developmental' genes is regulated by a mechanism that measures cell density relative to food supply, not by starvation per se.     

Release of cell wall peptides into culture medium by exponentially growing Escherichia coli.

Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation.