Early Exponential Growth Phase Research Articles

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa.

Treatment of Pseudomonas aeruginosa with metal ion chelators, especially ethylenediaminetetraacetic acid (EDTA), causes both release of protein-lipopolysaccharide complexes and cell death. We have examined the effect of EDTA on P. aeruginosa and found that EDTA does not induce the rapid solubilization of the peptidoglycan sacculus and complete lysis as previously thought; the decrease in optical density of cultures incubated with EDTA is primarily due to the loss of the outer membrane. Of the other potential solubilizers examined, only ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) resulted in some decrease in optical density. The lytic effect of EDTA on 12 strains of P. aeruginosa was examined and was found to vary greatly between strains; the sensitivity to EDTA varies from between 96% and 10% of the decrease in optical density resulting from incubation of cells with both EDTA and lysozyme. Sensitivity to EDTA is not constant during the growth of P. aeruginosa; in the early exponential phase of growth, cells treated with EDTA exhibit a 82% decrease in optical density after 30 min while in the stationary phase the optical density decreases by only 40%. Nucleic acids were observed to leak from cells following treatment with EDTA and this was greatly facilitated by DNase and RNase. The release of genetic material was much reduced when cells were incubated at 4 degrees C, supporting an enzymatic role in cell wall solubilization. We propose that only small areas of the sacculus become hydrolysed via specific peptidoglycan hydrolases, or autolysin(s), which are activated or de-regulated by EDTA.

Two o-Type Oxidases in Methylobacillus flagellatum KT

Two oxidases of the o-type in membranes of the methanol-grown obligate methylotroph Methylobacillus flagellatum KT were distinguished. For this purpose the kinetic analysis of the laser flash-induced optical absorbance changes of CO-oxidase complexes under reducing conditions was used. The ratio of these oxidases in membranes greatly depended on the phases of bacterial growth. One of the oxidases appeared to belong to the Escherichia colio-type oxidase family being more sensitive to KCN (Ki = 1 μM). It showed monophasic CO recombination kinetics with τ 25-30 ms and was expressed in the early exponential phase of growth. The other oxidase seemed to be similar to the Bacillus sp. FTU o-type oxidase being less sensitive to KCN (Ki = 6 μM), having three-phasic CO reassociation kinetics with τ 35-70 μs, 0.25-0.5 ms and 2-4 ms and dominating in the stationary growth phase. Pyridine haemochrome spectra showed haems A and D to be absent from the bacterial membranes.

Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1.

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.

Transcriptional regulation of Bacillus subtilis citrate synthase genes.

The Bacillus subtilis citrate synthase genes citA and citZ were repressed during early exponential growth phase in nutrient broth medium and were induced as cells reached the end of exponential phase. Both genes were also induced by treatment of cells with the drug decoyinine. After induction, the steady-state level of citZ mRNA was about five times higher than that of citA mRNA. At least some of the citZ transcripts read through into the isocitrate dehydrogenase (citC) gene. Transcription from an apparent promoter site located near the 3' end of the citZ gene also contributed to expression of citC. In minimal medium, citA transcription was about 6-fold lower when glucose was the sole carbon source than it was when succinate was the carbon source. Expression of the citZ gene was repressed 2-fold by glucose and 10-fold when glucose and glutamate were present simultaneously. This latter synergistic repression is similar to the effect of glucose and glutamate on steady-state citrate synthase enzyme activity. CitR, a protein of the LysR family, appeared to be a repressor of citA but not of citZ.

Biosynthesis of SOD by carrot hairy roots

Superoxide dismutase(SOD) was produced by carrot hairy roots. After increasing in the early exponential growth phase, SOD biosynthesis decreased as the growth rate of hairy roots declined. High sucrose concentration stimulated the SOD biosynthesis, while inhibiting the growth of hairy roots. Potential inducers were tested for the enhanced production of SOD. The addition of 0.3 mM CuSO4 to the broth showed about 6 fold increase in SOD biosynthesis compared to the results obtained with basic MS medium. Since CuSO4 in the broth inhibited the growth of hairy roots, the addition of CuSO4 to the broth at the late exponential growth phase led to a 12 times higher intracellular level of SOD compared to that in basic MS medium.

N remineralization in planktonic protozoa

Ammonium excretion rates of two marine planktonic protozoa (an oligotrichous ciliate, Strombidium sulcatum, and an aplastidic flagellate, Pseudobodo sp.) both isolated from oligotrophic Mediterranean waters were quantified with heat‐killed bacteria as the food source for the protozoans. Ammonium excretion rates ranged from 0.25 to 2 µg N mg DW−1 h−1 for the ciliate and from 2.8 to 8.5 for the phagotrophic flagellate. Maximum excretion rates took place during the early exponential growth phase, suggesting possible covariation of excretion rates with either protozoan body size or their growth rates or metabolic state. Comparison of these excretion and regeneration rates with those obtained in situ for oligotrophic waters (∼0–37 nmol liter−1 h−1) indicates that protozoa may account for >50% of the nutrient recycling in these waters.

MRNAs in the methanogenic archaeon Methanococcus vannielii: numbers, half-lives and processing.

Cells from the early exponential growth phase of cultures of the methanogenic archaeon Methanococcus vannielii have been shown to contain c. 180 transcripts of the mcrBDCGA (mcr) operon, c. 100 transcripts of the MvaL1,L10,L12 (Mva) operon, c. 8 transcripts of the argG gene and c. 1 transcript of the secY gene. These values decreased to c. 50 mcr transcripts, c. 30 Mva transcripts, c. 3 argG transcripts and < 1 secY transcript per cell as the cultures entered the stationary phase of growth. Addition of bromo-ethanesulphonate (BES) or removal of H2 inhibited growth and RNA synthesis in vivo and, at 37 degrees C in the presence of BES, the half-lives of the mcr, Mva, argG and secY transcripts were found to be 15 min, 30 min, 57 min and 7 min, respectively. Addition of puromycin, pseudomonic acid or virginiamycin also inhibited growth but did not inhibit transcription. In the presence of puromycin the half-lives of the mcr and Mva transcripts increased c. 4.6-fold and c. 3.5-fold, respectively, and there was a net accumulation of the Mva transcript. Addition of pseudomonic acid or virginiamycin also increased the half-life of the Mva transcript and also resulted in the accumulation of a second, shorter Mva transcript but did not increase the half-life of the mcr transcript. Transcription of the mcr operon was not stimulated by partial inhibition of methanogenesis.

Characterization of the 1,3- -glucan synthase of Aspergillus fumigatus

1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors--GTP, NaF, sucrose and EDTA--added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

Potential Application of LASER/Microbe Bioassay Technology for Determining Water-Soluble Vitamins in Foods

A microbiological technique was developed for quantitating niacin by determining microbial growth rates in response to the amount of vitamin available. Unlike the current official AOAC method, the new procedure for niacin measured the growth rates during the early exponential growth phase rather than during the stationary phase. Lactobacillus plantarum was used to determine niacin to a lower limit of 100 pg/mL. The assay time was approximately 6 h, compared with 16-24 h for the current AOAC method. The extent of microbial growth was determined by differential light scattering of a LASER beam. Multiple photodetectors were integrated with a computer system to collect and analyze the data. The use of differential light scattering to determine 8 water-soluble vitamins under stationary phase conditions demonstrated the potential application of the new technology for microorganisms and foods.

Regulation of muscarinic stimulation of norepinephrine release and PI hydrolysis during cell growth in PC12 cells

We have studied the relationships of cell growth to muscarinic stimulation of norepinephrine release and phosphoinositide hydrolysis in the rat pheochromocytoma PC12 cells. The ability of these cells to release norepinephrine in response to muscarinic agonists was maximal during the early phase of exponential growth, and then rapidly decreased to undetectable levels as the cells approached stationary phase. In contrast, muscarinic stimulation of phosphoinositide hydrolysis was low in the early exponential phase of growth, increased to a maximum during late exponential growth and then dramatically dropped in the stationary phase. The number of muscarinic receptors, as measured by antagonist-binding studies, also varied during cell growth with maximal levels at days 2 and 8, corresponding to the maxima in muscarinic-stimulated norepinephrine release and phosphoinositide hydrolysis, respectively.

Changes in Chemical Components and Energy Charge during Growth Cycle of Chattonella antiqua.

The changes in cellular content of adenosine triphosphate (ATP) in Chattonella antiqua (Hada) Ono (Raphidophyceae), a red tide microorganism, were determined during growth in laboratory culture using ESM-enriched seawater. Carbon, nitrogen, and phosphorus in the cells were also analyzed. The cellular content of ATP decreased within several hours of inoculation into fresh medium, then rapidly increased during the early exponential phase of growth to 20-30pg ATP cell-1. After the mid exponential phase ATP decreased and reached a constant level from the late exponential phase to the stationary phase at 5-10pg ATP cell-1. The decrease of ATP after inoculation seemed to be due to a lack of extracellular organic materials in the fresh medium. The addition of both low and high molecular fractions obtainned by dialysis from early stationary phase cultures of C. antiqua increased the cellular ATP content in the early exponential phase. The energy charge decreased from 0.63 to about 0.5 during the lag phase and recovered during the exponential phase. The lag phase in C. antiqua culture is the period of adaptation to fresh medium possibly by the production of extracellular organic materials at the expense of cellular ATP.

Modeling the population dynamics of baculovirus‐infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields

The insect cell-baculovirus model presented here is capable of simulating cell population dynamics, extracellular virion densities, and heterologous product titers in reasonable agreement with experimental data for a wide rang of multiplicities of infection (MOI) and times of infection. The model accounts for the infection of a single cell by multiple virions and the consequences on the time course of infection. The probability of infection by more than one virion was approximated using the Poisson distribution, which proved to be a refinement over second-order kinetics. The model tracks initiation and duration of important events in the progression of infected cell development (virus replication, recombinant protein synthesis, and cell lysis) for subpopulations delineated by the time and extent of their initial infection. The model suggests infection strategies, weighing the importance of MOI and infection time. Maximum product titers result from infection in the early exponential growth phase with low MOI.

Developmental expression of genes involved in conidiation and amino acid biosynthesis in Neurospora crassa

The levels of transcripts for Neurospora crassa genes concerned with cellular and metabolic functions changed dramatically at different stages of asexual development. Transcripts for some conidiation-related ( con) genes were present at high levels in conidiating cultures and in dormant conidia, but were absent or reduced during mycelial growth. Levels of some con transcripts increased transiently during conidial germination, while others disappeared. Transcripts for amino acid biosynthetic enzymes, ribosomal proteins, cytochrome oxidase subunits, histones, and other polypeptides important for cell growth were detected in newly formed conidia and were present at reduced levels in dormant conidia. Levels of these transcripts increased upon germination of wild-type conidia in minimal medium, reaching their highest levels during this stage or during the early phase of exponential growth. The increased transcription of amino acid biosynthetic genes observed during germination in minimal medium was not dependent on a functional cpc-1 gene. However, cpc-1, which encodes a DNA binding protein presumed to function as a transcriptional activator, was essential for increased expression of amino acid biosynthetic genes when amino acid starvation was imposed during germination or at any subsequent stage of mycelial growth.

Effect of carbon source and oxidative metabolism on secondary metabolism inStreptomyces thermoviolaceus

The effect of carbon source on a number of cultural parameters was investigated inStreptomyces thermoviolaceus. Glucose-grown cultures produced the antibiotic granaticin during pH-controlled growth in a fermenter. Biphasic growth occurred for all the carbon sources tested at 45°C, the inflexion of which occurred at a biomass density of between 0.5 and 0.6 gL−1 and which coincided with the onset of appearance of secondary metabolites. Maximum antibiotic production occurred in proline-grown cultures, which also had the slowest growth rates during the secondary phase of growth. Respiratory chain activity was probed by measuring NADH oxidation in membrane preparations exposed to a range of cyanide concentrations. Modulation of the terminal oxidase activity was apparent so that membranes prepared from cultures in the antibiotic-producing phase were less sensitive to KCN than those prepared from the early exponential phase of growth. The probable reason for this difference was the synthesis of cytochrome oxidased during later stages of growth. These changes in respiratory activity are discussed in relation to patterns of growth and timing of the appearance of secondary metabolites synthesised byS. thermoviolaceus.

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC50 of < 0.25 μg ml−1 to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g−1 (dry cells).

Bioproduction of [ 14C]deoxyphomenone, A fungistatic metabolite of the hyperparasite Dicyma pulvinata

Deoxyphomenone is a fungistatic sesquiterpene produced by the fungal hyperparasite Dicyma pulvinata. A study of the kinetics of the synthesis of this toxin was achieved by the use of [U- 14C]acetate. Labelled deoxyphomenone was detectable in the culture filtrate one hour after inoculation of the medium with spores of D. pulvinata. Optimum incorporation of radioactivity in the toxin occurred in the early phase of exponential growth. Consequently, maximum availability of deoxyphomenone is found in the young parts of the hyphae in zones where contact between the hyperparasite and parasite occur. This underlines the role which the toxin can play in early phases of hyperparasitism.

Two glycosylation patterns for a single protein (exoglucanase) in Saccharomyces cerevisiae

Exoglucanases (β-glucosidases) I and II secreted into the culture medium by Saccharomyces cerevisiae were purified from cell cultures harvested at the early exponential phase of growth in order to avoid contamination of the second by a new immunologically-related material. The amino acid composition of the purified enzymes was roughly the same. In addition, both exoglucanases exhibited an identical NH 2-terminal sequence (50 residues). These results confirm our previous results about the identity of the protein moieties of both enzymes. Exoglucanase I appears to arise by elongation of one or both short oligosaccharides present in enzyme II.

Two glycosylation patterns for a single protein (exoglucanase) in Saccharomyces cerevisiae

Exoglucanases (beta-glucosidases) I and II secreted into the culture medium by Saccharomyces cerevisiae were purified from cell cultures harvested at the early exponential phase of growth in order to avoid contamination of the second by a new immunologically-related material. The amino acid composition of the purified enzymes was roughly the same. In addition, both exoglucanases exhibited an identical NH2-terminal sequence (50 residues). These results confirm our previous results about the identity of the protein moieties of both enzymes. Exoglucanase I appears to arise by elongation of one or both short oligosaccharides present in enzyme II.

Optimized transformation by electroporation ofLactobacillus plantarum strains with plasmid vectors

Whole cell transformation ofLactobacillus plantarum CCM 1904 by electroporation was optimized. Pulse duration and electric field strength were shown to be important parameters: the optimum conditions were 12.5 kV/cm, a time-constant of 10 ms for an exponential decay waveform and 6.7 kV/cm applied during 2.5 ms for a square waveform. Transformation efficiency was increased if cells were cultivated on medium containing sorbitol and harvested during their early exponential growth phase: 8 × 10−4 transformants/μg pGK12 DNA per viable cell were obtained, with a survival rate of 10%–30% Cryotreatment by several freeze-and-thaw cycles decreased transformant yields. Transformation efficiency with different plasmids was studied and plasmid pGK12 was found to transformL. plantarum the most efficiently. Transformation by electroporation ofL. plantarum is strain dependent. The best results were obtained withL. plantarum NCIB 7220, giving 5 × 106 transformants/gmg plasmid pGK12 DNA.

Regulation of Activity of an ATP-Dependent Protease, Clp, by the Amount of a Subunit, ClpA, in the Growth of Escherichia coli Cells1

The activity of an ATP-dependent protease, Clp, was examined in Escherichia coli SG1110 (lon-) in various growth phases. The ATP-dependent proteolytic activity (Clp activity) in a crude extract of the cells changed with the growth phase. Cells in the early exponential growth phase showed the lowest activity, but then the activity increased dramatically with cell growth. The highest Clp activity was found in the cells in the late exponential and early stationary phases, however, the activity returned to the original level on prolonged culturing. These changes in Clp activity were closely correlated to the amount of one of the components of Clp, Clp A, which was quantitated immunochemically with antibodies against the Clp A protein. However, the amount of the other component of Clp, Clp P, did not change with the growth phase. These results suggest that the activity of Clp in the cells is regulated by the amount of Clp A in various growth phases. We next examined the effect of the cellular ATP level on Clp activity, because ATP is a cofactor for Clp protease in vitro. The addition of dinitrophenol (DNP) and sodium azide reduced the intracellular concentration of ATP, but had no effect on the Clp activity or the level of the Clp A protein when these drugs were added to the culture at the stationary phase. On the other hand, these drugs elevated both the Clp activity and the Clp A amount in exponentially growing cells, whose cellular ATP level was also reduced.(ABSTRACT TRUNCATED AT 250 WORDS)