Early Log Phase Cells Research Articles

Morphometry and stereology of the conversion of thin-walled yeasts to phase I yeast cells ofWangiella dermatitidis

Morphometric and stereological analyses of the conversion of thin-walled yeast cells to thick-walled phase I yeast cells of Wangiella dermatitidis were conducted over 15 days of growth. Log phase growth persisted for 48-60 hours and there was no loss in viability up to 15 days. Average cell volume dropped from 23 ^m3 on days 1 and 2 to 18 jiim3 on days 3 and 4, then increased to 38 jxm5 by day 15. Wide confidence limits for measurements of cell volume at each point indicated a very heterogeneous population. Wall thickness increased throughout the growth period with thickness increasing from 0.04 /um to 0.30 jj,m and the volume fraction from 0.10 to 0.24. Mitochondria and nuclei together made up less than 8% of the cell vol? ume [volume fraction of mitochondria, 0.025 (2.5%) and volume fraction of nuclei, 0.05 (5.0%)]. Volume fractions of both declined over time, as a result of increased cell volume from isotropic growth, although nuclei also may have become smaller. Glycogen was always present and increased to about 18% of the cell volume by day 4 but then decreased to 10% by day 15. Lipid was absent in early log phase cells but made up about 40% of the cell volume by day 15, and is presumably synthesized at the expense of glycogen and from residual sugar. Vacuoles, evident in early log cells (10-20% of the cell volume), declined rapidly and were rare after 8 days.

Effects of osmotic stress and growth stage on cellular pH and polyphosphate metabolism in Neurospora crassa as studied by 31P nuclear magnetic resonance spectroscopy

High-resolution 31P-NMR was employed to investigate the effects of growth stage and environmental osmolarity on changes of polyphosphate metabolism and intracellular pH in intact Neurospora crassa cells. Our study showed that changes of these parameters were growth-dependent. The ratio of polyphosphate to orthophosphate in vacuoles increased from 2.4 to 13.5 in N. crassa as cells grew from early log phase to stationary phase. Cytoplasmic pH and vacuolar pH changed, respectively, from 6.91 and 6.49 in early log phase cells to 7.25 and 6.84 in stationary phase cells. Hypoosmotic shock of N. crassa produced growth-dependent changes including: (i) a rapid hydrolysis of polyphosphate with a concomitant increase in the concentration of the cytoplasmic phosphate, (ii) an increase in cytoplasmic pH, and (iii) an increase in vacuolar pH. Early log phase cells produced the most dramatic response whereas the stationary phase cells appeared to be recalcitrant to the osmotic stress. Thus, 95% and 60% of polyphosphate in the early log phase and mid-log phase cells, respectively, disppeared in response to hypoosmotic shock, but little or no hydrolysis of polyphosphate occurred in stationary cells. The cytoplasmic pH and the vacuolar pH increased in response to hypoosmotic shock by 0.4 and 0.53 unit, respectively, in early log phase cells; and by 0.22 and 0.27 unit, respectively, in the mid-log phase cells. In contrast, hypoosmotic shock of the stationary phase cells did not cause any change in intracellular pH. The osmotic stress-induced polyphosphate hydrolysis and pH changes in early log and mid-log phase cells were reversible, suggesting that these changes were related environment osmolarity. Addition of polyamines or basic amino acids which are known to be sequestered in vacuoles did not affect polyphosphate metabolism.

Determination of cell-wall porosity by microscopy: walls of cultured cells and pollen tubes

In previous studies the porosity of the plant cell wall has been estimated to lie between 3.5 and 6 nm, depending on the cell type investigated and the method used. Morinda citrifolia cells from cultures at various stages of growth were incubated for 60 min with a colloidal gold sol (CGS) of size range 1.9-7.9 nm. Penetration of the cell wall by these gold particles was limited to the log phase of the culture growth cycle, with maximum uptake occurring in early log phase. The size of particles observed within the early log phase cells ranged from 2.5 to 5.7 nm. Since the cell wall was not, as in other methods, disrupted or altered, we conclude that the wall pores are up to 5.7 nm mean diameter at this early stage, but decrease rapidly as the culture ages

ENHANCING CELL RECEPTIVITY TO GENE TRANSFER BY ELECTRIC DISCHARGE PARTICLE ACCELERATION

Successful recovery of plants transformed by direct gene transfer techniques relies on 3 factors: 1) a regenerable cell/tissue culture system, 2) a foreign DNA delivery system which can be fine tuned, and 3) a cell population receptive to the transfer and integration of foreign DNA into its genome. Cell receptivity to foreign DNA incorporation is being determined by bombarding poplar cells with gold particles coated with plasmid DNA containing a CaMV35s pro–meted β-glucuronidase chimeric gene construct. Histochemical GUS expression assays conducted after bombardment show that early log phase cells are most receptive with a transient transformation rate of 0.26% compared to 0.08–0.04% for mid log and stationary phase cells. Furthermore, cell receptivity in early log phase cells is decreased when cytokinin is removed from the culture medium for 3 growth cycles prior to bombardment. This response suggests that plant growth regulators may be used to enhance cell receptivity along with cell synchronizing agents.

The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors.

Escherichia coli mutator mutD5 is the most potent mutator known. The mutD5 mutation resides in the dnaQ gene encoding the proofreading exonuclease of DNA polymerase III holoenzyme. It has recently been shown that the extreme mutability of this strain results, in addition to a proofreading defect, from a defect in mutH, L, S-encoded postreplicational DNA mismatch repair. The following measurements of the mismatch-repair capacity of mutD5 cells demonstrate that this mismatch-repair defect is not structural, but transient. mutD5 cells in early log phase are as deficient in mismatch repair as mutL cells, but they become as proficient as wild-type cells in late log phase. Second, arrest of chromosomal replication in a mutD5-dnaA(Ts) strain at a nonpermissive temperature restores mismatch repair, even from the early log phase of growth. Third, transformation of mutD5 strains with multicopy plasmids expressing the mutH or mutL gene restores mismatch repair, even in rapidly growing cells. These observations suggest that the mismatch-repair deficiency of mutD strains results from a saturation of the mutHLS-mismatch-repair system by an excess of primary DNA replication errors due to the proofreading defect.

A temporally expressed gene from Schwanniomyces alluvius and detection of homologous sequences in other yeasts

The boundaries of a temporally expressed gene ( EG1) located on a 6.5-kb HindIII chromosomal DNA fragment of the yeast Schwanniomyces alluvius have been defmed by cDNA hybridization and S1 nuclease mapping. The sequence of this gene reveals a 474-bp open reading frame (158 aa) and a TATA-like sequence 100 bp upstream from the first start codon. The encoded protein bears little resemblance with other known yeast structural proteins, including histones. A portion of the 3' untranslated region shows 50% identity with a consensus repetitive sequence (B2) found in the mouse genome. Repetitive sequences are also located in both 5' and 3' untranslated regions. Genomic DNA analysis indicated that related sequences were detected at two loci in Schw. alluvius and one locus in Saccharomyces cerevisiae. In contrast to the continuous transcription pattern of the ADE1 genes of Schw. alluvius and S. cerevisiae, the 0.8-kb transcripts of EG1 in these yeasts were most abundant in cells in early-log phase of growth and absent in cells of late-log phase, suggesting discontinuous synthesis.

Expression of an antigen in strains of Salmonella typhimurium which reacts with antibodies to cholera toxin.

Six strains of Salmonella typhimurium (W118, TML, SL1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1-4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.

Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii

Rhizobium trifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4'-dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4'-dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10 M. Cells in other growth phases were generally unresponsive. A 10-fold molar excess of umbelliferone to 7,4'-dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commercially-obtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7-hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.

Lysis of Streptococcus sanguis by an extracellular enzyme from the bacterium Streptococcus mutans from human dental plaque

The ability of crude extracellular enzyme produced by the oral bacterium Streptococcus mutans AL7-1 to lyse living cells of Streptococcus sanguis ATCC 10556, 10557 and 10558 was examined. This enzyme showed lytic activity of living cells and cell walls of only Strep. sanguis ATCC 10558 strain and severed at random the long chains of this strain of living cells. Early log phase cells of this strain were more sensitive to this lytic enzyme than were late-log phase cells. In view of these results, the relationship between this lytic enzyme from Strep. mutans and a decrease in the number of serotype III strains of Strep. sanguis in dental plaque is discussed.

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent Mrs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS–PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile. These strains were divided into two groups on the basis of the wall protein composition: one containing Mr 45 000–47 000 and 32 000 proteins and the other containing Mr 42 000 and 38 000 proteins.

Demonstration and preliminary characterization of a regular array in the cell wall ofClostridium difficile

The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent Mrs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile. These strains were divided into two groups on the basis of the wall protein composition: one containing Mr 45 000–47 000 and 32 000 proteins and the other containing Mr 42 000 and 38 000 proteins.

Demonstration of age-dependent capsular material on Pasteurella haemolytica serotype 1.

Extracellular capsular material was demonstrated on early log-phase cells of Pasteurella haemolytica serotype 1 by the fluorescent-antibody and several capsular staining techniques. The presence of this material was shown to be age dependent. Wide capsules were demonstrable on cells from 2- to 12-h cultures, whereas cells from 16- to 22-h cultures had very little cell-associated capsular material. The Maneval technique most clearly demonstrated the presence of capsules on cells from young (6-h) cultures when compared with other capsule staining techniques.

Decomposition of Casein by Caseobacter polymorphus Strain 253

Caseobacter polymorphus strain 253 belongs to the nonorange coryneforms predominant on Limburger and similar soft cheeses and is thought to be involved in ripening processes by way of casein decomposition. During growth on sodium caseinate in an otherwise mineral salt medium, endopeptidase, aminopeptidase, and iminopeptidase were produced by the organism. Endopeptidase was extracellular and optimally active at pH 6.5 to 7.0 and 60°C. Its formation roughly parallels growth of the bacterium on sodium caseinate and is inhibited by amino acids, in particular glutamic acid, and by utilizable carbon sources, particularly lactose and glycerol. Aminopeptidase was optimally active at pH 7.35 and 40°C and iminopeptidase at pH 7.45 and 50°C. Their activities increased only slightly during the log phase but sharply at the onset of the stationary phase. Their extracellular log-phase activities were elevated sharply by glutamic acid but iminopeptidase only by a mixture of amino acids. These three proteases are regulated independently.Ultrasonic treatment of early log-phase cells stimulated their aminopeptidase activity. The L-proline-4-nitroanilide-cleaving activity of intact cells was similar to the activity of sonicated cells. Particularly in early log-phase cultures there were only minor extracellular aminopeptidase and iminopeptidase activities (5 to 15% of the cell-bound activities). At least part of the caseinate is entering the cells as peptides instead of single amino acids.

Modulation of a Cell Surface Glycoprotein in Yeast: Acid Phosphatase

Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed. Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different. This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme. Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell.

Sensitivity and permeability of the anthramycin producing organism Streptomyces refuineus to anthramycin and structurally related antibiotics.

Streptomyces refuineus, the microorganism which produces the DNA reactive antibiotic anthramycin, has shown to possess a quite specific mechanism to survive and grow in the presence of this antibiotic. Stationary phase cells are insensitive to anthramycin since the antibiotic is prevented form entering these cells. However, cells in early log phase are inhibited by concentrations of anthramycin that are later produced by these same cells. Significantly, sibiromycin, a closely related antibiotic, is taken up by cells of S. refuineus independent of the age of the culture. Anthramycin reacts in vitro equally as well as DNA isolated from S. refuineus and other procaryotic and eucaryotic cells. When S. refuineus has reached the production phase the anthramycin is probably biosynthesized outside the cell membrane which also becomes specifically impermeable to anthramycin.

Differentiation promoting factors induced in Acanthamoeba by inhibitors of mitochondrial macromolecule synthesis

Acanthamoeba castellanii (line OS4) differentiates into a cyst when treated with mitochondrial inhibitors berenil, ethidium bromide, erythromycin, and chloramphenicol. The process is a function of cell density at the time of inhibition: Early log phase cells yield 0–20% cysts, whereas mid-log phase cells yield up to 55, 65, or 95% cysts when treated with chloramphenicol or erythromycin, ethidium bromide, or berenil, respectively. Medium from encysting mid-log phase cells contains an encystment enhancing activity (EEA) which, when transferred to low-density early log phase cultures, causes high levels of encystment. EEA has a nominal molecular weight of between 1000 and 10,000. It is resistant to nucleases, proteases, amylase, boiling, and 1 N NaOH, 25°C, but is inactivated by snake venom phosphodiesterase, autoclaving, and acidic or harsher alkaline treatments. Other lines, OS1 and OS5, which do not encyst well at any cell density in response to the four inhibitors or to EEA, nevertheless produce EEAs to which OS4 can respond. OS4 has been adapted to growth in a chemically defined medium; when starved for glucose and acetate, the cells produce a phosphodiesterase-sensitive EEA. A similar activity can be obtained from mitochondria treated with berenil in vitro. Several lines of evidence suggest that EEAs from the various sources are the same. We propose that they may be of mitochondrial origin and may act as developmental effectors under conditions unfavorable for cell growth.

Effect of growth conditions on yield and heme content of Vitreoscilla.

Vitreoscilla, a gliding bacterium in the Beggiatoaceae, is an obligate aerobe in which cytochrome o functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of Vitreoscilla cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome o content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for Vitreoscilla, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.

The regulation of DNA replication during senescence and rejuvenation of Dunaliella tertiolecta by factors other than available metabolic energy

The unicellular green alga, Dunaliella tertiolecta undergoes a 4-fold reduction in DNA while progressing from early to late log phase of culture. During the period when the reduction in DNA occurs, the cells continue to divide at the maximal rate. Pulse labelling indicates little incorporation of [ 3H]thymidine into DNA during late log phase. Stationary phase cultures diluted with fresh medium undergo a lag period during which there is a 4-fold increase in DNA and a rapid incorporation of [ 3H]thymidine into DNA into DNA before division. The evidence indicates that the differences in the levels of DNA are not attributable to tetraploidy, multinucleated cells or a high level of redundancy of G-C-rich satellite DNA in early log phase cells. During stationary phase there is an increase in cellular starch and a decrease in free nucleotides. Electron microscopy reveals that stationary phase cells are distorted by many granules proven to be starch by their susceptibility to amylase treatment. The production of starch by stationary phase cells indicates that DNA replication does not cease because of a deficit of metabolic energy but because of some direct function of culture density.

The influence of the condition of cells and medium on production of polyhedra of Autographa californica nuclear polyhedrosis virus in vitro

A culture of an established cell line of Trichoplusia ni (TN-368) was infected with the nuclear polyhedrosis virus of Autographa californica (ACNPV) at different stages of growth. After 50 hr of growth (early log phase), production of polyhedral inclusion bodies (PIBs) declined rapidly as cell number increased. Medium taken from cultures in the later stages of growth suppressed PIB production in early log-phase cells. Cultures infected with ACNPV and inoculated into fresh medium at high cell concentrations produced relatively fewer PIBs than those inoculated at lower concentrations. The depressive effect of utilized medium is suggested to be the result of exhaustion of a precursor rather than accumulation of an inhibitor.

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[14C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[14C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.