Effects of pressure on cell morphology and cell division of lactic acid bacteria

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.Communicated by K. Horikoshi     

Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells ofArabidopsis thaliana transformed with green-fluorescent-protein gene constructGFP-MBD

Summary Arabidopsis thaliana plants were transformed withGFPMBD (J. Marc etal., Plant Cell 10: 1927–1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35SGFP-MBD plants grown on kanamycincontaining media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the wo processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more efined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubuleorienting factor(s) may be sensitive to growth acceleration, rather than growth per se.     

High temperature enhances cytotoxicity of mercury (HgCl2) on Hela S3 cells

The combined effect of mercury (HgCl₂) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S₃ cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2° and 41.2°C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC₅₀ values for DNA and RNA synthesis but not protein synthesis, at 41.2°C, were significantly lower than those at 37.2°C (P<0.05,P<0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures.     

Extracellular factors responsible for the adaptation ofPseudomonas fluorescens batch cultures to unfavorable conditions

The culture liquid filtrate of an exponential-phasePseudomonas fluorescens batch culture added to anotherP. fluorescens culture at the moment of inoculation was found (1) to prevent or diminish cell adsorption of the flask walls, (2) to enhance the intensity of cell respiration, (3) to shorten the period of adaptation of LB-grown cells to growth in glucose-containing mineral M9 medium, (4) to stimulate bacterial growth at supraoptimum temperature (36°C) and pH values (4.8 and 9.2), and (5) to decrease the death rate of bacteria at the supraoptimum growth temperature. These results were interpreted as indicating thatP. fluorescens cultures produce two types of regulatory exometabolites similar to those revealed earlier inEscherichia coli andBacillus subtilis cultures: the direct-action adaptogenic factorX ₁ capable of increasing bacterial resistance to unfavorable growth conditions (temperature and pH) and factor of accelerated adaptation to new media. Both factors are presumably low-molecular-weight hydrophilic nonprotein compounds.     

Morphometry and growth of three <Emphasis Type="Italic">Synechococcus</Emphasis>-like picoplanktic cyanobacteria at different culture conditions

Three phycocyanin-rich strains of Synechococcus-like picoplanktic cyanobacteria, isolated from the plankton of Czech oligotrophic to eutrophic freshwater reservoirs, were investigated in crossed gradients of light and temperature and in combination with two different culture media (BG-11 and WC). The strains exhibited similar growth and reproduction patterns and displayed overlapping ranges of cell size (1.5 × 0.8 μm) under standardized laboratory conditions (18 μmol m⁻² s⁻¹; 20°C). However, strains behavior differed in the crossed gradients. All strains preferred BG-11 medium, where also remarkable size changes could be observed. Length, width, cell abundance and growth rate of two strains were positively correlated with temperature and nutrients, whereas the impact of light intensity was insignificant. Maximum cell elongation (involution cells up to 19 μm) occurred in two strains only in BG-11 medium at highest temperature (28°C) and highest irradiance (53 μmol m⁻² s⁻¹). Cell dimensions in WC medium were constant under most conditions given. The third strain was influenced by all three factors, from which light and nutrients played pivotal role. The length of the lag-phase for all strains appeared to be temperature dependent (negative correlation). Despite the fact that the cell volume in all strains increased more than five times under the lowest light and low temperature (6 μmol m⁻² s⁻¹, <15°C) in both media, the length/width ratio remained unchanged. The strains differed in the degree of cell enlargement and cell division symmetry as well as in optimum temperature and light dependence. Based on this experimental work two strains could be identified as Synechococcus sp. and one as Cyanobium sp., which can be used as a support for the following genetical analyses.     

Why get big in the cold? Towards a solution to a life-history puzzle

The temperature–size rule (TSR), which states that body size increases at lower developmental temperatures, appears to be a near-universal law for ectotherms. Although recent studies seem to suggest that the TSR might be adaptive, the underlying developmental mechanisms are thus far largely unknown. Here, we investigate temperature effects on life-history traits, behaviour and physiology in the copper butterfly Lycaena tityrus in order to disentangle the mechanistic basis for the above rule. In L. tityrus the larger body size produced at a lower temperature was proximately due to a greater increase in mass, which was caused by both behavioural and physiological mechanisms: a much-increased food intake and a higher efficiency in converting ingested food into body matter. These mechanisms, combined with temperature-induced changes at the cellular level, may provide general explanations for the TSR. Body fat and protein content increased in butterflies reared at the higher temperature, indicating favourable growth conditions. As predicted from protandry theory, males showed reduced development times, caused by higher growth rates compared to females. The latter was itself related to a higher daily food consumption, while the total food consumption (due to the females’ longer developmental period) and assimilation was higher in females and may underly the sexual body size dimorphism.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Effects of cell entrapment on growth rate and metabolic activity of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on the growth rate and metabolic activity of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different cell entrapment media (alginate, carrageenan and polyvinyl alcohol) and three cell-to-matrix ratios (0.1%, 0.2% and 0.6%, w v⁻¹) were examined. Representative species of betaproteobacteria were Alcaligenes faecalis and Comamonas testosteroni whereas Pseudomonas putida was a gammaproteobacteria species studied. Free (non-entrapped) cells were included in the study for comparative purpose. Results indicated that the entrapment, type of entrapment media, and cell-to-matrix ratio had significant effects on the growth and metabolic activity of major groups of bacteria in wastewater treatment. Polyvinyl alcohol entrapped cells had the highest specific growth and specific substrate utilization rates. Increase of cell-to-matrix ratio (from 0.1% to 0.2% or 0.6%) did not improve the specific growth and specific substrate utilization rates. The relative performances provided by different entrapment media of the three species studied were quite consistent. This study showed that the suitable choices of entrapment media and cell-to-matrix ratio are important but similar for major groups of bacteria in wastewater treatment.     

Effect of temperature on the size of Escherichia coli cells.

The distributions of cell volumes of steady-state Escherichia coli ML30G cultures at various temperatures were measured. For cultures in a minimal medium, the distributions were indistinguishable at several temperatures between 15 and 30 C; at higher temperatures the cells were slightly smaller, and at lower temperatures they were slightly larger. For cultures in a complex medium, the cells were slightly larger at both high and low temperatures of growth. An abrupt change of temperature within the middle range led to a transient change in the distribution of cell volume, suggesting that the size of dividing cells is well regulated. No synchrony of division was induced by a change in temperature.     

Effects of polyunsaturated fatty acids on the growth and differentiation of the cellular slime mould, Dictyostelium discoideum

Cells of Dictyostelium discoideum grown on media containing polyunsaturated fatty acid (PUFA) exhibit impaired differentiation when placed on a solid surface in the absence of all nutrients. Differentiation is inhibited at all temperatures, and although the inhibition is somewhat less pronounced at low temperatures, there is neither a lowering of the optimum temperature of aggregation nor a reversal of inhibition at low temperatures. Furthermore, although the in vitro aggregation of vegetative cells and the reaggregation of dispersed aggregation-phase cells are markedly temperature dependent, PUFA supplementation does not markedly influence this dependence. These data are not consistent with the hypothesis that impaired differentiation is due to increased plasma membrane fluidity. PUFA had no adverse effect on cell growth at temperatures at or below the optimum growth temperature, 22 °C. At 25 °C, however, there was considerable inhibition and at 27 °C growth was completely eliminated in the presence of PUFA.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.     

The response of white clover (Trifolium repens L.) seedlings to spring root temperatures: The relative roles of the plant and the Rhizobium bacteria

Three experiments are reported which examine the relative roles of host and Rhizobium genotypes as factors limiting clover (Trifolium repens L.) growth at low soil temperatures.In the first experiment un-nodulated clover and perennial ryegrass (Lolium perenne L.) were grown with non-limiting nitrate at root temperatures of 8, 10 and 12°C. The ryegrass had substantially better relative growth rates (RGR) than the clover with the biggest difference occurring at 8°C. Alterations in growth rate with temperature were more marked in clover than in ryegrass but the latter still produced several times more dry matter than clover at each temperature.In the subsequent experiments clover nodulated with different strains of rhizobia was grown with and without non-limiting additions of nitrate at root temperatures of 9, 12 and 15°C. Plants receiving nitrate generally produced more dry matter than those dependent upon Rhizobium for nitrogen but differences in yield between these treatments did not alter with temperature. This suggests that limitations imposed by nitrogen fixation are similar at both high and low temperatures. Indeed, there was some evidence that nitrogen limitations were rather more pronounced at the highest temperature. The first experiment clearly demonstrated that the clover genotype makes particularly poor use of nitrate at low root temperatures when compared to its common companion perennial ryegrass.It can be concluded that improvements in spring growth of clover will rest largely with alterations to the plant genotype and its ability to use combined nitrogen for growth at lower temperatures rather than with changes in rhizobia or any symbiotic characters.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Effects of temperature and light on cyst germination and germinated cell survival of the noxious raphidophyte Heterosigma akashiwo

The effects of temperature and light on the germination of Heterosigma akashiwo cysts were examined using bottom sediments collected from Hakata Bay, Japan. In a suspension of mixed sediment and seawater in the temperature range of 5–30 °C, motile cells emerged within 3 weeks, but at ≤12 °C the cell numbers were markedly lower and the emergence of motile cells delayed. When suspension samples incubated at various temperatures were moved to 20 °C and incubated, only a few additional motile cells emerged. The number of motile cells germinated in the dark was significantly lower than under light conditions. When suspension samples incubated in the dark were exposed to light, only a few additional motile cells emerged. These results indicate that the initiation of germination in Heterosigma cysts suspended in seawater is not dependent on temperature and light conditions, although the speed of the germination process is affected by temperature, and cell survival just after germination is strongly affected by temperature and light.     

MEL-47, a novel protein required for early cell divisions in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50–80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.