Early Logarithmic Growth Phase Research Articles

In vitro activity of vancomycin against enterococci.

The in vitro activity of vancomycin against 40 clinical isolates of enterococci was determined by a macro-tube dilution method and by quantitative killing curve procedures employing the standard medium of our department, i.e. a filtered ox broth. An attempt to remove the influence of technical factors on the MBC determination was made by using an inoculum in the early logarithmic growth phase and ensuring the exposure of all the organisms to the antibiotic. Vancomycin showed a good inhibitory activity for the enterococci tested (MIC90 of 1.6 micrograms/ml, 3.1 micrograms/ml and 1.6 micrograms/ml for S. faecalis, S. faecium and S. durans, respectively), but no bactericidal effect could be demonstrated as measured by the MBCs (greater than 100 micrograms/ml) and killing curve procedures.

Characterization of an estrogen-binding protein in the yeast Candida albicans.

An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.

On the presence of calmodulin-like protein in mycobacteria

A heat-stable factor, antigenically cross-reacting with calmodulin, was found mainly in the soluble fraction of Mycobacterium smegmatis ATTC 14468 and Mycobacterium bovis BCG 14 004 001. Maximum amounts of calmodulin were found in the early logarithmic phase of growth. The cell-free extract of M. smegmatis did not stimulate, rather inhibited cAMP phosphodiesterase due to the presence of certain endogenous inhibitory protein(s). However, the cAMP phosphodiesterase stimulation could be observed, after the removal of inhibitor(s). This is the first report indicating the presence of calmodulin in mycobacteria, a member of the prokaryotes.

On the presence of calmodulin-like protein in mycobacteria

A heat-stable factor, antigenically cross-reacting with calmodulin, was found mainly in the soluble fraction of Mycobacterium smegmatis ATTC 14468 and Mycobacterium bovis BCG 14 004 001. Maximum amounts of calmodulin were found in the early logarithmic phase of growth. The cell-free extract of M. smegmatis did not stimulate, rather inhibited cAMP phosphodiesterase due to the presence of certain endogenous inhibitory protein(s). However, the cAMP phosphodiesterase stimulation could be observed, after the removal of inhibitor(s). This is the first report indicating the presence of calmodulin in mycobacteria, a member of the prokaryotes.

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm −2), medium (1000 cells cm −2), and high (10,000 cells cm −2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60 % of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.

The influence of aluminum on the cell-size distribution of two green algae

Cell-size distribution of batch cultures of two green algae Monoraphidium and Stichococcus were studied in relation to aluminium exposure. The cell size of Monoraphidium was more differentiated than that of Stichoccus. The distributions changed during the growth period but differed between the two algal species; Monorphidium appeared bimodal before cell division, whereas Stichococcus showed a one-peak distribution pattern. Changes in mean cell size for the two algal populations were similar during growth in batch cultures, the cells grew larger before division. Mean cell size decreased during the logarithmic growth phase; during the stationary growth phase a slight increase reappeared. Both species displayed cell decomposition when exposed to aluminum. The surviving cells grew larger than the control cells when aluminum was added to the medium. The effects of aluminum oncentration on cell size differed during the growth phases for the two algal cultures. The algal populations seemed to be most sensitive during the lag and the early logarithmic growth phases.

Action spectrum of the photoattractant response of Halobacterium halobium in early logarithmic growth phase and the role of sensory rhodopsin

Photoattractant response was measured in a relatively carotenoid-poor strain derived from the mutant of Halobacterium halobium that lacks both bacteriorhodopsin and halorhodopsin (strain Flx 3). No photoattractant response was observed in the cells at logarithmic growth stage, coinciding with the fact that there was no sensory rhodopsin in membrane fraction prepared from the cells in logarithmic growth stage as measured by flash photholysis experiment. When all- trans-retinal was added to the cell suspension or the membrane suspension, the phototactic activity or the photocycling due to sensory rhodopsin appeared rapidly. This indicates that apoprotein of sensory rhodopsin had been formed in the cells at the growth stage, and suggests that the photoattractant response was mediated by sensory rhodopsin. The action spectrum of the photoattractant response resembled sensory rhodopsin absorption at wavelengths than 600 nm, but was distorted at shorter wavelengths by the photorepellent system that was found recently.

Carbon Assimilation in Carrot Cells in Liquid Culture

Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with d-[(14)C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified ;energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools.

Effect of some nutritional and environmental factors on extracellular protease production by Pseudomonas SP.B-25

SUMMARV Fortification of the 1 % tryptone basal medium with 0.25 % yeast extract resulted in 2.6-fold increase in protease production by Pseudomonas sp.B-25. Tryptone yeast extract in the medium could not be substituted by other equivalent media ingredients. Of the various inorganic salts incorporated in the modified basal medium Cl% tryptone + 0.25 % yeast extract), addition of 0.2 % potassium phosphate salts elicited maximum enzyme production. However, none of the fermentable sugars tried as supplements to the formulated tryptone-yeast extract-phosphate (TYEP) medium proved useful. Maximum protease production was observed in TYEP medium at pH 7.0 when inoculated with 2.5 % of 24 hr old broth culture and incubating at 22° C for 48 h on a gyratory shaker. The protease production by Pseudomonas sp.B-25 was found to be initiated in the early logarithmic growth phase and reached the maximum when the culture was in the decline phase of growth.

PZ-peptidase activities of some black-pigmented Bacteroides species

Bacteroides gingivalis (both W 83 and ATCC 33277 strains), B. asaccharolyticus (ATCC 25260), and B. melaninogenicus (ATCC 25845) produced hydrolytic activity on the synthetic peptide 4-phenylazobenzyl-oxycarbonyl-L-prolyl-L-leucyl-glycyl-prolyl-D-arginine (PZ peptide) during their early logarithmic phase of growth. The PZ-peptidase activity of the ATCC 25260 strain was the highest with an initial rate of reaction at 0.54 nmol PZ-pro-leu/h per 108 cells. Relatively low levels of PZ peptidase were produced by the pathogenic W 83 cells. In all the strains tested, no further increase in PZ-peptidase activity was detected after the late logarithmic phase, even when the cultures lysed. The result of the protease test seems to indicate that the PZ-peptidase activity of both oral strains of B. gingivalis is collagenase, while that of B. asaccharolyticus may be a mixture of protease and collagenase enzymes.

Differential regulation of the duplicated isocytochrome c genes in yeast.

The two unlinked genes CYC1 and CYC7 encode iso-1-cytochrome c and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae. An examination of the steady-state level of CYC1 and CYC7 mRNAs in normal and mutant strains grown under different conditions, along with previous results of apoprotein levels, demonstrate that CYC1 and CYC7 have similar and different modes of regulation. Both CYC1 and CYC7 mRNAs are diminished after anaerobic growth. In contrast, CYC1 mRNA but not CYC7 mRNA is decreased by heme deficiency in hem1 mutants. Although both CYC1 and CYC7 mRNAs are substantially lowered after growth in glucose medium, there is a difference in the kinetics of glucose derepression. CYC1 mRNA levels rise in the early logarithmic phase of growth before complete exhaustion of glucose, whereas CYC7 mRNA levels rise in the late logarithmic phase when the level of CYC1 mRNA has plateaued. For a brief period before cessation of growth, the level of CYC7 mRNA attains a level corresponding to the high derepressed level of CYC1 mRNA. The high amount of CYC7 mRNA is surprising because iso-2-cytochrome c constitutes only 5% of the total cytochrome c complement in derepressed cells. We suggest that iso-2-cytochrome c has the potential to comprise a major proportion of cytochrome c under certain physiologic conditions that have not been experimentally defined. The cyc3 mutant, which lacks the ability to attach heme groups to apocytochromes c, contains both CYC1 and CYC7 mRNAs in normal amounts. Yet, cyc3 mutants contain only apoiso-2-cytochrome c and not apoiso-1-cytochrome c. The lack of accumulation of apoiso-1-cytochrome c in cyc3 mutants, which contain CYC1 mRNA, suggests that apoiso-1-cytochrome c is extensively regulated by a post-transcriptional process.

Effect of alcohols on the production of lytic enzyme by a bacterium isolated from coastal waters.

The suppression of the production of lytic enzyme was investigated using alcohols as membrane-modifying agents. At the concentrations which growth was not suppressed, the production of lytic enzyme by strain V 37 was inhibited by n-alcohols having a range of 8 to 14 carbon atoms, phenethyl alcohol and benzyl alcohol, respectively. The formation of extracellular lytic enzyme by the cells from the lag phase to the early logarithmic phase of growth was most susceptible to membrane-modifying agents such as octanol and phenethyl alcohol.

Secretion of an exo-poly-.ALPHA.-D-galacturonosidase by Erwinia carotovora during the early logarithmic growth phase.

Secretion of an exo-poly-.ALPHA.-D-galacturonosidase by Erwinia carotovora during the early logarithmic growth phase.

Increased production of alpha-amylase by Bacillus amyloliquefaciens in the presence of glycine.

The production of alpha-amylase by Bacillus amyloliquefaciens increased by a factor of 300 when glycine was added to a chemically defined simple medium at the early-logarithmic phase of growth. Glycine was not metabolized to a significant extent under the conditions used, but it considerably prevented the lowering of the pH of the culture.

Nitrogen fixation and ammonia switch-off in the photosynthetic bacterium Rhodopseudomonas viridis.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.

Changes in Embryonic Cell Frequencies in the Germinative and Immature Regions of Hymenolepis citelli during Development

This study traces the growth rate of Hymenolepis citelli and correlates such growth to the relative frequency of the only mitotically active cell found in the germinative and immature regions of the cestode. The abundance of this cell, termed the germinative cell, was very high during the early logarithmic growth phase of the cestode and decreased just prior to patency to a level that was maintained until day 90 postinfection. An increase in germinative cell frequency was observed at day 90. It is postulated that this increase may reflect a developmental change which has occurred in the cestode following patency.

Extracellular production of proteins by microorganisms. Part V. Stimulatory effect of inhibitors of cell wall synthesis on protein production by Bacillus brevis.

Mutants of Bacillus brevis No. 47 that grew in synthetic media containing a high concentration of ammonium sulfate were stable and had high protein production. Among various antibiotics tested, inhibitors of cell wall synthesis, such as bacitracin or β-lactam antibiotics, were effective in greatly increasing the accumulation of exoproteins. When 60μg/ml of bacitracin was added to the culture at the early logarithmic growth phase, about 9mg/ml of proteins was produced. Such a protein yield was estimated to be nearly maximum from a given amount of glucose. Alterations in cell wall components were found in cells grown in the presence of bacitracin. Possible relationships between cell wall structure and protein production were discussed.

Production of vitamin B12, thiamine, and biotin by freshwater phytoplankton.

B group vitamin requirements and production were examined in axenic cultures of several freshwater phytoplankters which were isolated from the water of a eutrophic lake, Lake Kojima. All the phytoplankters used, Scenedesmus sp., S. abundance, Chlorella sp. (A), Chlorella sp. (B), Chlamydomonas sp., Caraeria sp., and Chroococcus sp., were found to have no vitamin requirements and to produce considerable amounts of vitamin B12, thiamine, and biotin in the culture media as the age of the culture increased. The vitamins produced as well as the production rate of the vitamins varied with both the species of the phytoplankters and the stages of the growth. Generally the B group vitamins were secreted increasingly with the cell multiplication. The amounts of the B group vitamins produced per ml of medium per day during the early logarithmic phase of growth were generally more abundant than those during the stationary phase. On the other hand, the amounts of vitamins produced per cell per day did not vary regularly with the different stages of the growth. These values were mostly 10-8-10-7 pg/cell/day for vitamin B12, 10-6-10-5 pg/cell/day for thiamine, and 10-5-10-4 pg/cell/day for biotin. From the present results, it is suggested that some species of the phytoplankters may significantly contribute to the production of the dissolved B group vitamins found abundantly in the waters of the lake.

Mitomycin C stimulates production of a toxin in Shigella species that causes morphological changes in Chinese hamster ovary cells

Shigella dysenteriae produced a toxin that caused morphological changes in Chinese hamster ovary cells which was different from the neurotoxin (Y. Takeda, K. Okamoto, and T. Miwatani, Infect. Immun. 18:546--548, 1977). It was found that the toxin was produced not only by S. dysenteriae but also by S. flexneri, S. boydii, and S. sonnei. The production of the toxin was stimulated by the addition of 1 microgram of mitomycin C per ml to the culture in the early logarithmic growth phase.

Clinical bacteriological studies on the beta-lactamase produced by Bacteroides fragilis (author's transl)

Strains of Bacteroides fragilis, being highly resistant against some β-lactam antibiotics (Penicillin, Sulbenicillin, Cephalothin) especially by the hyperinoculation of 1010 bacterial cells per ml, made strains of drug-sensitive enteric bacteria grow aerobically on the drug-containing media.These sensitive strains, mixed either washed cells or culture filtrate of resistant B. fragilis, grew on the media containing the drugs in 100μg/ml or higher concentration.However, when the inoculum size of 1010 cells/ml of B. fragilis in mixed culture was reduced to 106 cells/ml, the growth of the sensitive strains on the drug-containig media became disproportionaly poor. In addition, no strain of the enteric bacteria obtained from the mixed culture could be subcultured on the same drug-containing media.From these experiments, it appeared that this growth of the enteric bacteria was not resulted from the transfer of R plasmid from the drug-resistant B. fragilis, but from the inactivation of the antibiotics by the resistant organisms.The ability of B. fragilis to inactivate β-lactam antibiotics was detected in the supernatant of washed and sonicated cell-suspension as well as culture filtrate at early logarithmic phase of bacterial growth. As this inactivating ability was especially remarkable to cephalothin, the essential nature of this ability might be due to cephalosporinase of B. fragilis.Among the strains of B. fragilis which did not show the drug-inactivating ability in the mixed culture experiments, some strains were as highly resistant as drug-inactivating strains on the sensitivity tests carried out with 106 cells per ml. However, they were neither so resistant on the tests with 1010 cells/ml, nor they produced any cephalosporinase in their culture filtrate though a certain enzymatic activity was demonstrated in their cells.Amount of resistant anaerobes and their cephalosporinase or β-lactamase activity should always be considered at the chemotherapy in which any β-lactam antibiotics is applied.