Exponentially-growing Cultures Research Articles

Nutrient recycling facilitates long-term stability of marine microbial phototroph-heterotroph interactions.

Biological interactions underpin the functioning of marine ecosystems, be it via competition, predation, mutualism, or symbiosis processes. Microbial phototroph-heterotroph interactions propel the engine that results in the biogeochemical cycling of individual elements and are critical for understanding and modelling global ocean processes. Unfortunately, studies thus far have focused on exponentially-growing cultures in nutrient-rich media, meaning knowledge of such interactions under in situ conditions is rudimentary at best. Here, we performed long-term phototroph-heterotroph co-culture experiments under nutrient-amended and natural seawater conditions which showed that it is not the concentration of nutrients but rather their circulation that maintains a stable interaction and a dynamic system. Using the Synechococcus-Roseobacter interaction as a model phototroph-heterotroph case study we show that whilst Synechococcus is highly specialised for carrying out photosynthesis and carbon-fixation it relies on the heterotroph to re-mineralise the inevitably leaked organic matter making nutrients circulate in a mutualistic system. In this sense we challenge the general belief that marine phototrophs and heterotrophs compete for the same scarce nutrients and niche space, but instead suggest these organisms more likely benefit from each other because of their different levels of specialization and complementarity within long-term stable-state systems.

Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the invivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different transcriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.

Growth and Starvation of a Strain of Klebsiella pneumoniae Isolated from a Brazilian Oil Formation

The changes that occur in cell size and shape during experimental starvation and resuscitation of bacteria may find practical application in the biotechnological processes used in microbial enhanced oil-recovery (MEOR), in in situ bioremediation and in the creation of biobarriers. In the work described here, the aim was to observe certain aspects of the response of a strain of Klebsiella pneumoniae, isolated from oil producer wells belonging to PETROBRAS (National Oil Company of Brazil), to starvation and growth under various culture conditions. Cells of K. pneumoniae were taken from an exponentially-growing culture, washed and suspended in phosphate-buffered saline solution, without nutrients, where they remained for 91 days. Aliquots were withdrawn periodically, to perform viable and total cell counts and measure the optical density and cell dimensions. The starved cells were resuspended in sodium citrate medium, where they recuperated and exhibited a typical growth curve. The results indicate that this strain is a viable option for future trials on the transport and growth of microorganisms inside porous media, aimed at possible applications in MEOR.

Development of biochemical methods to estimate the subcellular impact of uranium exposure on Chlamydomonas reinhardtii

This work aims at determining early effects of uranium on the green microalga Chlamydomonas reinhardtii. First, the global effect on growth rate inhibition of exponentially-growing cultures was assessed on favourable conditions for uranium bioavailability (e.g. pH=5), EC50-24hrs equals roughly to 150 nM whatever the uranium isotopic composition considered (depleted U or 233 U). Then, the sensitivity of different parameters representative of (i) oxidative stress (GSH/[GSH + 0.5 GSSG] ratio) (ii) metal detoxifying (phytochelatins production) and (iii) photosynthetic activity (chlorophyll fluorescence) was tested. Setting assay of different forms of glutathione and phytochelatins by HPLC was firstly optimised with cadmium-contaminated cells. This assay completed by chlorophyll fluorescence and algal growth was subsequently applied on samples contaminated by 150 nM of depleted uranium or 233 U. No phytochelatin was produced in our experimental conditions. No difference of GSH/[GSH + 0.5 GSSG] ratio was shown between control and contaminated algae. This result suggests that the algae could be stressed before contamination due to culture condition. Chlorophyll fluorescence measurement showed photosynthetic activity inhibition after 24 hrs, in the same way for depleted uranium and 233 U. Thus, the effect observed on the photosynthetic activity could be mainly attributed to the chemical toxicity of the metal.

Lactose-induced cell death of ?-galactosidase mutants in

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Detection and analysis by dual‐laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli

Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual-laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A-T- and G-C-rich regions of DNA, respectively. An exponentially-growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol-fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty-five minutes post-infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA-containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 +/- 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual-laser flow cytometry can be used to study viral DNA inside the bacterial host.

Effects of the carotenoid-inhibiting herbicide, fluridone, on Oscillatoria agardhii Gomont (cyanobacteria)

The effects of the pyridinone herbicide, fluridone, on the growth, pigment content and composition, and photosynthetic capability of Oscillatoria agardhii Gomont were investigated. Batch-culture flasks were inoculated with equal amounts of culture volume from an exponentially-growing culture and subjected to fluridone concentrations ranging from 0 to 100 μg. 1- i. Culture populations were allowed to grow without interruption for 96 h after which populations were analyzed. Biomass, and chlorophyll a and total carotenoid contents decreased with increasing fluridone concentration. No change was observed in C-phycocyanin content. Within the lipophilic pigment fraction, decreases in the relative abundances of myxoxanthophyll, zeaxanthin, β-carotene, and an unidentified, minor carotenoid caused corresponding increases in the relative abundance of chlorophyll a. The light-limited slope, the maximum light-saturated rate of photosynthetic O 2 evolution, and the irradiance value at onset of light saturation decreased with increasing fluridone concentration. Inhibition of growth and chlorophyll content was considered a secondary response attributable to the photo-oxidation of chlorophyll molecules. Inhibition of the photosynthetic efficiency and maximum were attributed to reduced carotenoid content.

Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation.

PC12 cells were manipulated in such a way as to permit the study of differentiation-specific responses independently from proliferative responses. Cells were starved for serum then exposed to nerve growth factor (NGF) or serum. Following addition of serum, cells incorporated thymidine in a synchronous manner. Subsequent to the wave of DNA synthesis, the cell number increased approximately two-fold. Addition of NGF to serum-starved cultures had no measurable effect on either parameter. Neurite outgrowth was more rapid and extensive and appearance of Na+ channels, measured as saxitoxin binding sites, more rapid than when NGF was added to exponentially-growing cells. Epidermal growth factor receptors were heterologously down-regulated by NGF with similar kinetics under both conditions. Induction of the proto-oncogene c-fos by NGF was also greater in the serum-starved cells than in exponentially-growing cultures. These results indicated that serum starvation resulted in synchronisation of the cultures and that NGF action may be cell cycle-specific. Analysis of the cellular response to NGF at different times during the cell cycle showed that c-fos was induced in the G1 phase but not in S or G2. Fluorescence-activated cell sorter analysis demonstrated that addition of NGF to exponentially-growing cells, resulted in their accumulation in a G1-like state. With regard to the study of the mechanism of NGF action, these results illustrate that measurements of NGF effects on specific components in the signal transduction pathway may be confounded by the use of exponentially-growing cultures.

FLOW CYTOMETRIC ANALYSIS OF INTRACELLULAR HEMATOPORPHYRIN DERIVATIVE IN HUMAN TUMOR CELLS AND MULTICELLULAR SPHEROIDS

Flow cytometry (FCM) has been used to investigate the intracellular fluorescence of hematoporphyrin derivative (HPD) in monolayer and spheroid cultures of WiDr cells. For exponentially-growing monolayer cultures mean cellular fluorescence was directly proportion to the external HPD levels in the range 5-100 micrograms ml-1 (r = 0.99). Heterogeneity of cellular fluorescence was quantified by determining the ratio of the fluorescence value below which were observed values for 98% of the cell population compared to the fluorescence value for 2%. In exponentially-growing cultures, decreasing levels of HPD in the medium led to an increase in the 98:2% ratio, i.e. an increase in heterogeneity of intracellular drug levels. The growth of cells as multicellular spheroids confers a spheroid-size-dependent resistance to photodynamic treatment. With increasing spheroid size (100, 250, 500, 750 and 1000 microns diam.) there was a decrease in mean intracellular HPD levels and a large linear increase in the 98:2% ratio (r = 0.94).

Requirement for mevalonate in cycling cells: quantitative and temporal aspects.

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.

Influence of a 1400-gauss Magnetic Field on the Radiosensitivity and Recovery of EMT6 Cellsin Vitro

EMT6 mouse mammary tumour cells in vitro were exposed to an almost uniform 1400-gauss magnetic field during or after irradiation with 120 kV X-rays. Exposure of unirradiated control cultures to this field for up to 48 hours did not alter the viability or growth of the cells. Exposure of exponentially-growing cultures to the magnetic field during irradiation did not alter the survival curve. Exposure of exponentially-growing culturesto the magnetic field between two doses of 500 rad of X-rays did not alter significantly the pattern or magnitude of the recovery from sub-lethal damage. Exposure of plateau phase cultures to the magnetic field after irradiation did not alter significantly the pattern or magnitude of recovery from potentially-lethal damage. The effects of a short exposure to an almost uniform magnetic field of this strength on the production and repair of radiation damage appear to be minimal in this system.

Monomer Addition as a Mechanism of Forming Peptide Cross-Links in the Cell-Wall Peptidoglycan of Streptococcus faecalis ATCC 9790

The relative amounts of radioactively labelled disaccharide-peptide monomers and peptide-cross-linked dimers and trimers found in the peptidoglycan of Streptococcus faecalis ATCC 9790 were compared to the relative amounts to be expected from two different polymerization mechanisms (random condensation and monomer addition). Data from continuously-labeled, exponentially-growing cells are consistent with a monomer addition cross-linking process, not with a random condensation cross-linking mechanism. This conclusion was supported by data obtained from analyses of cells labeled during valine starvation (and wall thickening), recovery from valine starvation, and pulse and pulse-chase labeling of walls from exponentially-growing cultures.

Studies on the acid-soluble nucleotide pool in Escherichia coli: IV. Effects of hydroxyurea

Addition of hydroxyurea to exponentially-growing cultures of Escherichia coli resulted in a concentration-dependent decrease in the acid-soluble deoxyribonucleoside triphosphate pools of the cells. At the same time a corresponding relative decrease in the rate of DNA synthesis was observed, suggesting that the limiting factor for DNA synthesis in hydroxyurea-treated cultures of E. coli is the size of the precursor pool. In contrast, the ribonucleoside triphosphate pools did not change significantly during treatment with the drug.

THE EFFECT OF IONIZING RADIATION ON A MARINE PSEUDOMONAS SP. II. THE TOTAL EFFECTS OF POPULATIONS IN DIFFERENT GROWTH-PHASE.

SummaryInactivation of Pseudomonas sp. No. 128 was found to be independent of concentration up to a critical level of 2 × 108 org./ml.The highest sensitivity was noted in the exponentially-growing cultures, and the highest resistance in cultures in the negative accelerating growth-period (0·5 and 50·0 per cent survival, respectively, for 10·0 krad established immediately after irradiation). Resistance was essentially the same during lag and late declining periods (1·0 per cent survival for the standard dose used).Exponentially-growing cultures showed a continuously increasing inactivation on delay of the plating after irradiation (from 0·5 to 0·005 per cent survival at 10·0 krad). No such effects were noted for cultures in late declining or maximum stationary growth-periods.Survival-curves for samples taken from maximum stationary and declining growth-periods were exponential. A cumulative type of survival-curve was noted for organisms from the lag and logarithmic periods of growth.