Stimulating effect of pancreatic DNase on Bacillus subtilis growth as a function of the physiologic properties of the inoculation culture

The stimulating effect of pancreatic DNAse on Bacillus subtilis growth was studied in relation to the content of "slowly growing" cells in the inoculation culture in the phase of decelerated growth. Three cell fractions of B. subtilis were obtained using the stepwise separation of the population in terms of buoyant density in the phase of decelerated growth. In contrast to fractions II and III, fraction I contained cells with decelerated growth, competent, permeable to exogenous DNAase I, and sensitive to the action of this enzyme. The faster growth of bacterial cells in fraction I was shown to be associated with the shorter lag period of these cells having a longer generation time.     

Effect of pancreatic DNAse on Bacillus subtilis multiplication depending on the physiological state of the culture

The effect of pancreatic DNAase on the growth of Bacillus subtilis, the synthesis of DNA, RNA and protein in the cells was studied as a function of their physiological state. The cells were found to be sensitive to the action of DNAase at the end of the linear growth phase, which coincided with their transition into the competence state and with their ability to adsorb the enzyme on the cell surface.     

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria

Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.     

New Chromatographic Form of Phenylalanine Transfer Ribonucleic Acid from Escherichia coli Growing Exponentially in a Low-Phosphate Medium

Reversed-phase chromatography has been used to detect the presence of a new form of phenylalanyl-transfer ribonucleic acid (Phe-tRNA) from Escherichia coli growing exponentially in media containing low but nonlimiting levels of inorganic phosphate. The amount of this extra Phe-tRNA form is greatest in slowly growing cells (0.8 generations/h), and becomes negligible in media supporting a rapid growth rate (2.14 generations/h).     

Change in the activity of DNAse I inhibitor in the mouse spleen on the administration of a synthetic polyanion

Synthetic polyanion pyran (copolimer of divinyl ether and maleic anhydride) injected to mice increases the titre of antibody to sheep red blood cells as well as the activity of serum DNAase I and DNAase I splenic inhibitor. Simultaneously a growth of the spleen weight takes place. A possible role of the DNAase I inhibitor system in the mechanism of the adjuvant action of synthetic polyanion is discussed.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

Permeability of Escherichia coli K-12 cells to exogenous nucleodepolymerases

The permeability of Escherichia coli cells for exogenous nucleodepolymerases has been studied by an immunoenzyme method. The enzyme ability to penetrate through the bacterial outer membrane and cell wall after 20 min of incubation with culture cells of delayed growth phase has been found.     

Inhibitory action of a non-metabolizable fatty acid on the growth of Escherichia coli: role of metabolism and outer membrane integrity.

The inhibitory action of decanoic acid on both Escherichia coli K-12/154 (normal lipopolysaccharide) and E. coli RC59 (defective lipopolysaccharide) was studied. A correlation was found between the doubling time of E. coli 154 growing in different media and the lethal effect of 0.4% decanoic acid on this bacterium. Decanoic acid (0.4%) exerted a lytic action on glucose-starved and NaN3-inhibited cells of E. coli 154 and RC59. Exponentially growing cultures of both strains were not affected by the addition of 0.4% methyldecanoate, but cells of E. coli RC59 reaching the stationary phase were attacked by that compound. A bactericidal action of 0.4% methyldecanoate on exponential E. coli 154 and RC59 was observed when sodium azide was also present in the media. Concentrations lower than 0.01% methyldecanoate had a lytic effect on spheroplasts from E. coli 154 and RC59. These results indicate that the inhibitory action of a non-metabolizable fatty acid on E. coli depends on the cellular metabolic activity and the outer membrane integrity.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134–144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G₁ phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G₁ phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G₁- and S-phase cells, respectively. The G₁ cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G₁ nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

Regulation of DNA chain elongation in SV3T3 cells in relation to growth rate.

Regulation of DNA synthesis was investigated in SV40 transformed 3T3 cells exhibiting variable growth rates and residence times in S phase when cultured in the presence of different serum concentrations. Pulse-labeled DNA was chased into large molecular weight material in vivo much more slowly in slowly growing cells than in cells growing at the normal rate. Consistent with this, the joining of short (less than 10 S) chains to form long (greater than 10 S) chains by whole cell lysate system in vitro was greatly impaired in slowly growing cells compared to controls. Thus the lengthening of S phase in SV3T3 cells growing slowly in low serum is reflected in a reduced rate of DNA chain elongation. The presence of cycloheximide during chase in vivo reduced the rate of conversion of pulse-labeled molecules into large molecular weight DNA in both slowly growing and normally growing cells.     

Existence of two chalone-like substances in intestinal extract from the adult newt, inhibiting embryonic intestinal cell proliferation.

The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and 3-H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G1 phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G2 phase. The cells delayed in the G2 phase escape from inhibition but the cells delayed in the G1 phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G1 phase are indefinitely delayed in this phase and possibly differentiate.     

Distribution of bacterial growth activity in flow-chamber biofilms.

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

The effect of guanidine hydrochloride on the conformation of bovine pancreatic DNAase I as measured with circular dichroism

The denaturation of bovine pancreatic DNAase I (EC 3.1.21.1) by guanidine hydrochloride (GdnHCl) has been investigated with circular dichroism in the presence and absence of 1 mM Ca2+ at the wavelength region of 210-240 nm at 12.25 and 36 degree C. The change of the molar ellipticity at 220 nm by GdnHCl titration showed cooperative transition at each temperature and the midpoints of the titrations occurred near 2 M GdnHCl. At each temperature, the denaturation of DNAase I in the presence of 1 mM Ca2+ occurred a little slowly as compared with that in the absence of Ca2+. This suggests that 1 mM Ca2+ can to some extent stabilize the secondary structure of DNAase I against GdnHCl denaturation. The apparent free energy for the denaturation of DNAase I obtained by GdnHCl titration was calculated as 9.3 +/- 0.3 kcal/mol and 8.9 +/- 0.2 kcal/mol at 25 degree C in the presence and absence of 1 mM Ca2+, respectively. The possible regions for the alpha -helix and beta -structure of DNAase I were predicted from the amino acid sequence by probability calculation of Chou, P.Y. and Fasman, G.D., Adv. Enzymol. 47, 45-148. The characteristic feature is that the NH2-terminal half of DNAase I is rich in beta -structure and the COOH-terminal half contains mainly alpha -helix.     

Control of gluconeogenic growth by pps and pck in Escherichia coli.

It is well-known that Escherichia coli grows more slowly on gluconeogenic carbon sources than on glucose. This phenomenon has been attributed to either energy or monomer limitation. To investigate this problem further, we varied the expression levels of pck, encoding phosphoenolpyruvate carboxykinase (Pck), and pps, encoding phosphoenolpyruvate synthase (Pps). We found that the growth rates of E. coli in minimal medium supplemented with succinate and with pyruvate are limited by the levels of Pck and Pps, respectively. Optimal overexpression of pck or pps increases the unrestricted growth rates on succinate and on pyruvate, respectively, to the same level attained by the wild-type growth rate on glycerol. Since Pps is needed to supply precursors for biosyntheses, we conclude that E. coli growing on pyruvate is limited by monomer supply. However, because pck is required both for biosyntheses and catabolism for cells growing on succinate, it is possible that growth on succinate is limited by both monomer and energy supplies. The growth yield with respect to oxygen remains approximately constant, even though the overproduction of these enzymes enhances gluconeogenic growth. It appears that the constant yield for oxygen is characteristic of efficient growth on a particular substrate and that the yield is already optimal for wild-type strains. Further increases in either Pck or Pps above the optimal levels become growth inhibitory, and the growth yield for oxygen is reduced, indicating less efficient growth.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

The quantitative determination of the DNAse activity in Staphylococcus aureus isolated from monkeys

A new, cheaper and more sensitive method for the quantitative determination of DNAase produced by S. aureus is described. The method permits the determination of DNAase activity in a wider range of titers. The method is based on the detection of the depolymerizing action of staphylococcal nuclease on DNA dyed with ethidium bromide. In this work 22 S. aureus strains isolated from monkeys and 12 strains isolated from humans have been used. The amount of produced by these strains has been determined. The DNAase results of this determination have shown that among S. aureus strains isolated from monkeys and humans the occurrence of strains with both high and low DNAase activity can be observed.