Biosynthesis of RNA polymerase in Escherichia coli VI. Distribution of RNA polymerase subunits between nucleoid and cytoplasm.

The distribution of DNA-dependent RNA polymerase in Escherichia coli was analysed by measuring enzyme subunits in nucleoid (folded chromosome) and cytoplasm. Two independent methods, two-dimensional polyacrylamide gel electrophoresis of total proteins and sodium dodecyl sulphate/polyacrylamide gel electrophoresis of antibody precipitates, gave essentially the same results; with wild-type cells growing at a doubling time of 70 minutes, about two-thirds of the core enzyme but little σ subunit are present in the nucleoid. Pulse-chase experiments indicated that the distribution of the pulse-labelled proteins was at equilibrium within 1·5 minutes for β′, 5 minutes for β, and 15 minutes for α subunit. This order of appearance of the newly synthesized core subunits into the nueleoid is in good agreement with that into complete enzyme structure. This finding, together with the known sequence of subunit assembly (2α → α₂ → α₂β → α₂ββ′ → E), indicates that the assembly of RNA polymerase takes place in the cytoplasm. In concert with the conclusion, the amounts of pulse-labelled subunits in the cytoplasm of temperature-sensitive assembly defective mutants coincide well with those of intermediate subassemblies accumulated in the mutant cells. However, it is not known if the premature core is activated in cytoplasm prior to binding to the nucleoid or shortly after association with the nucleoid.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Cooperativity in viral fusion

A simple model for membrane fusion mediated by vial spike glycoproteins is presented. The viral proteins are considered to be allosteric proteins that undergo concerted conformational transitions when they bind the ligand. The ligand in this case is H⁺. The effect of the conformational transition is to bring membranes together and induce their fusion. An equation is derived for the dependence of fusion rates on ligand concentration, for a given dissociation constant (K _d), equilibrium constant for the conformational change (L), and number of cooperating subunits (n). Curves generated by this equation provide a reasonable fit to data on the rates of fusion of Vesicular Stomatitis virus with cells for a pK _d of 6.3,L=1000 andn=6.     

Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in Escherichia coli

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.     

CO2 efflux under different grazing managements on subalpine meadows of Shangri-La, Northwest Yunnan Province, China

Soil carbon stored on the Tibetan Plateau appears to be stable under current temperature, but it may be sensitive to global warming. In addition, different grazing systems may alter carbon emission from subalpine meadow ecosystems in this region. Using a chamber-closed dynamic technique, we measured ecosystem respiration (ER) and soil respiration (SR) rates with an infrared gas analyzer on a perennial grazing meadow (PM) and a seasonal grazing meadow (SM) of Shangri-La in the Hengduan Mountain area. Both PM and SM showed strong unimodal seasonal variations, with the highest rates in July and the lowest in January. Significant diurnal variations in respiration were also observed on PM, affected mainly by air and soil temperatures, with the highest rates at 14:00 and the lowest before dawn. Both ER and SR rates were higher on PM than on SM from June to October, suggesting that the higher grazing pressure on PM increased respiration rates on subalpine meadows. The exponential model F = ae^{bT<,/sup> of soil temperature (T) explained the variation in respiration better than the model of soil moisture (W) (R2 = 0.50–0.78, P < 0.0001), while the multiple model F = aebT<,/sup>Wc gave better simulations than did single-factor models (R2 = 0.56–0.89, P < 0.0001). Soil respiration was the major component of ER, accounting for 63.0%–92.7% and 47.5%–96.4% of ER on PM and SM, respectively. Aboveground plant respiration varied with grass growth. During the peak growing season, total ecosystem respiration may be dominated by this above-ground component. Long-term (annual) Q₁₀ values were about twice as large as short-term (one day) Q₁₀. Q₁₀ at different time scales may be controlled by different ecological processes. The SM had a lower long-term Q₁₀ than did the PM, suggesting that under increased temperature, soil carbon may be more stable with reduced grazing pressure.}     

Estimation of Noise-Free Variance to Measure Heterogeneity

Variance is a statistical parameter used to characterize heterogeneity or variability in data sets. However, measurements commonly include noise, as random errors superimposed to the actual value, which may substantially increase the variance compared to a noise-free data set. Our aim was to develop and validate a method to estimate noise-free spatial heterogeneity of pulmonary perfusion using dynamic positron emission tomography (PET) scans. On theoretical grounds, we demonstrate a linear relationship between the total variance of a data set derived from averages of n multiple measurements, and the reciprocal of n. Using multiple measurements with varying n yields estimates of the linear relationship including the noise-free variance as the constant parameter. In PET images, n is proportional to the number of registered decay events, and the variance of the image is typically normalized by the square of its mean value yielding a coefficient of variation squared (CV ²). The method was evaluated with a Jaszczak phantom as reference spatial heterogeneity (CV_r ²) for comparison with our estimate of noise-free or ‘true’ heterogeneity (CV _t ²). We found that CV _t ² was only 5.4% higher than CV _r ². Additional evaluations were conducted on 38 PET scans of pulmonary perfusion using ¹³NN-saline injection. The mean CV _t ² was 0.10 (range: 0.03–0.30), while the mean CV ² including noise was 0.24 (range: 0.10–0.59). CV _t ² was in average 41.5% of the CV ² measured including noise (range: 17.8–71.2%). The reproducibility of CV _t ² was evaluated using three repeated PET scans from five subjects. Individual CV _t ² were within 16% of each subject''s mean and paired t-tests revealed no difference among the results from the three consecutive PET scans. In conclusion, our method provides reliable noise-free estimates of CV _t ² in PET scans, and may be useful for similar statistical problems in experimental data.     

Estimating genetic and environmental components of variance using sexual and clonal Artemia

Reproductive and life span traits were measured for two obligately parthenogenetic (Artemia parthenogenetica) and three sexual (two A. franciscana and one A. sinica) brine shrimp populations. For each population, clonal lineages or single mating pairs were followed through one life cycle. The relative contributions of environmental and genetic components to total phenotypic variation for 10 life-history traits in response to environmental stress (0, 10, 25 ppb Cu) were estimated. Within treatment variation (CV_W) was 39% higher for sexual populations than parthenogenetic populations, with significant (p<0.05) differences in total number of offspring and number of nauplii. CV_A (the change in variance due to rearing in different environments), when averaged for all traits and all populations, increased variability by 9.9%. CV_A was 44.2% higher for sexual than parthenogenetic populations, with significant differences in number of broods, total number of offspring, and number of nauplii. The average genetic component of variation for the 10 traits was 23.44%, ranging from 5.26% for number of cysts to 44.87% for number of nauplii. For all traits, the environmental component of variance is greater than the genetic component measured, but every trait has a genetic component, which can potentially be acted upon by selection.     

Associations between leaf developmental stability,variability, canalization,and phenotypic plasticity in Abutilon theophrasti

Developmental stability, canalization, and phenotypic plasticity are the most common sources of phenotypic variation, yet comparative studies investigating the relationships between these sources, specifically in plants, are lacking. To investigate the relationships among developmental stability or instability, developmental variability, canalization, and plasticity in plants, we conducted a field experiment with Abutilon theophrasti, by subjecting plants to three densities under infertile vs. fertile soil conditions. We measured the leaf width (leaf size) and calculated fluctuating asymmetry (FA), coefficient of variation within and among individuals (CV_intra and CV_inter), and plasticity (PI_rel) in leaf size at days 30, 50, and 70 of plant growth, to analyze the correlations among these variables in response to density and soil conditions, at each of or across all growth stages. Results showed increased density led to lower leaf FA, CV_intra, and PI_rel and higher CV_inter in fertile soil. A positive correlation between FA and PI_rel occurred in infertile soil, while correlations between CV_inter and PI_rel and between CV_inter and CV_intra were negative at high density and/or in fertile soil, with nonsignificant correlations among them in other cases. Results suggested the complexity of responses of developmental instability, variability, and canalization in leaf size, as well as their relationships, which depend on the strength of stresses. Intense aboveground competition that accelerates the decrease in leaf size (leading to lower plasticity) will be more likely to reduce developmental instability, variability, and canalization in leaf size. Increased developmental instability and intra‐ and interindividual variability should be advantageous and facilitate adaptive plasticity in less stressful conditions; thus, they are more likely to positively correlate with plasticity, whereas developmental stability and canalization with lower developmental variability should be beneficial for stabilizing plant performance in more stressful conditions, where they tend to have more negative correlations with plasticity.     

Photochemical Apparatus Organization in Anacystis nidulans (Cyanophyceae) : Effect of CO(2) Concentration during Cell Growth

Anacystis nidulans cells grown under high (3%) CO₂ partial pressure have greater phycocyanin to chlorophyll ratio (Phc/Chl) relative to cells grown under low (0.2%) CO₂ tension (Eley (1971) Plant Cell Physiol 12: 311-316). Absorbance difference spectrophotometry of A. nidulans thylakoid membranes in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions of the spectrum reveal photosystem II/photosystem I (PSII/PSI) reaction center ratio (RCII/RCI) changes that parallel those of Phc/Chl. For cells growing under 3% CO₂, the Phc/Chl ratio was 0.48 and RCII/RCI = 0.40. At 0.2% CO₂, Phc/Chl = 0.38 and RCII/RCI = 0.24. Excitation of intact cells at 620 nm sensitized RCII at a rate approximately 20 times faster than that of RCI, suggesting that Phc excitation is delivered to RCII only. In the presence of DCMU, excitation at 620 nm induced single exponential RCII photoconversion kinetics, suggesting a one-to-one structural-functional correspondance between phycobilisome and PSII complex in the thylakoid membrane. Therefore, phycobilisomes may serve as microscopic markers for the presence of PSII in the photosynthetic membrane of A. nidulans. Neither the size of individual phycobilisomes nor the Chl light-harvesting antenna of PSI changed under the two different CO₂ tensions during cell growth. Our results are compatible with the hypothesis that, at low CO₂ concentrations, the greater relative amounts of PSI present may facilitate greater rates of ATP synthesis via cyclic electron flow. The additional ATP may be required for the active uptake of CO₂ under such conditions.     

ON THE RELATION BETWEEN LITTER SIZE,BIRTH WEIGHT,AND RATE OF GROWTH,IN MICE

We have been concerned with the connection between size of litter and weight of litter at birth, especially in mice. The weight at birth represents, it is to be presumed (at least in mice, and for certain other cases), the weight at a particular developmental stage. The connection between number in litter (N) and weight of litter (W) has been interpreted as due to the partition of nourishment between mother and young, and on an equal basis among the several embryos of a litter. The "heterogonic" relationship which the data exhibit between N and W shows that the constant K, defined by log W = K log N + const., is independent of the species, and has an essentially constant value (0.85±) in all multiparous mammals; it is therefore regarded as a partition coefficient. In the case of power function relationships between masses of components of a single individual, the respective "drawing powers" of the several organs are diverse, and diverse magnitudes of K are encountered. With developing embryos, the intrinsic drawing powers of the tissues concerned in embryos and mothers are in each case of the same general character, at least among mammals; the constancy of K reflects this. A parallel for the case as it appears in the consideration of relative growth rates of organs in a single individual, and in which the varying magnitudes of the heterogonic growth constant K are presumed to reflect diverse drawing powers of the respective tissues, would be given by intrauterine growth of a litter containing individuals with diverse capacities for growth, —that is, individuals differing genetically with respect to the factors determining the magnitudes of w ₁. We have been dealing with the growth of litters in inbred strains. It is to be presumed that in the case of the growth of a litter containing two categories of individuals so far as concerns intrinsic drawing powers with respect to the nourishment provided by the mother, it would be possible to investigate the way in which K is open to modification. Although difficult, from the standpoint of classifying the individual young, it would appear to be distinctly worth while to make such an experiment, and we have planned it for the future. It is pointed out that for genetic purposes the ideal weight of a litter of 1 is obtainable from a series of measurements of N and W, free from disturbances affecting the apparent value of this quantity as observed in single births. This weight of an ideal litter of 1 should be employed to disentangle the effects of heterosis and fertility factors from those having to do with individual weight at birth. During the suckling period the relation ΔW/W = K (ΔN/N) is maintained for young mice, but with modifications in the case of small and large suckling litters due to (1) the time course of milk yield, and (2) the effect of litter size upon this. It is shown that a growth curve can be obtained for an ideal litter of I, under the condition of milk supply that on each day the mother is able to provide a constant fractional increase of milk for each additional young mouse in the litter. The rate of growth then adheres to the time curve of capacity for production of milk.     

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.     

Novel histone-like DNA-binding proteins in the nucleoid from the acidothermophillic archaebacterium Sulfolobus acidocaldarius that protect DNA against thermal denaturation

DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase T_m of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [³H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C).     

Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar⁻¹ and second⁻¹ (Cot_½ 0.24 molar second) under conditions equivalent to 180 millimolar Na⁺ and 60°C. Given the 92 × 10⁵ dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.     

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.     

Effect of Growth Rate and Starvation-Survival on Cellular DNA, RNA, and Protein of a Psychrophilic Marine Bacterium

DNA, RNA, and protein concentrations from starved ANT-300 cell populations grown at different growth rates fluctuated corresponding to the three stages of starvation-survival on total and viable cell bases. During stage 1 of starvation-survival, two to three peaks in the concentration levels for all three macromolecules were characteristic. During stage 2, DNA per total cell dropped to between 4.2 and 8.3% of the original amount for all of the cell populations examined, and it stabilized throughout stage 3. The decrease in DNA per cell was also observed in electron micrographs of cellular DNA in unstarved compared with starved cells. The fluctuations of RNA and protein per total cell concentrations observed during stage 2 coincided in all cases, except for the cells from dilution rate (D) = 0.015 h⁻¹. This ANT-300 cell population showed a decrease in RNA per total cell to only 29.2% and an increase in protein to 129.7% of the original amount after 98 days of starvation. During stage 3, DNA, RNA, and protein concentrations per total cell also stabilized to continuous levels. Cells from the faster-growth-rate cell populations of D = 0.170 h⁻¹ and batch culture had elevated protein per total cell concentrations, which remained primarily residual during the starvation period. Starved cells from D = 0.015 h⁻¹ had estimated nucleoid and cell volumes of 0.018 and 0.05 μm³, respectively, yielding a nucleoid volume/cell volume ratio of 0.40. We consider these data to indicate that slow-growth-rate cells are better adapted for starvation-survival than their faster-growth-rate counterparts.     

Effects of controlled irrigation on water potential,nitrogen uptake and biomass production in Dalbergia sissoo seedlings

Biomass production, pattern of nodulation, nutrient uptake, net photosynthetic rate (P_n), leaf temperature (T_leaf), leaf nitrate reductase (NR) activity and free proline of Dalbergia sissoo seedlings planted in containers with 120 kg soil were studied under different water stress levels to assess the productive potential of the species in dry areas. Seedlings were irrigated at 20 mm (W₁), 14 mm (W₂), 10 mm (W₃), 8 mm (W₄) throughout the experimental period to maintain the respective treatment up to the lowest soil water content of 7.43%, 5.64%, 4.30% and 3.23%, respectively. There was a treatment (W₅) in which seedling were irrigated once to −0.03 MPa and left without re-irrigation. Decreased irrigation level resulted in lowering of leaf water potential (LWP), net photosynthetic rate (P_n), total number of root nodules and nodule dry mass and nitrogen uptake in the seedling. P_n, leaf nitrate reductase (NR) activity and seedling biomass were highest in W₁ indicating a positive relations of NR activity with CO₂ assimilation and biomass production. The decrease in P_n, leaf NR activity and LWP was sharp at W₃ onwards. Monthly changes in the values of P_n, T_leaf and NR activity indicate environmental effect on these physiological variables. Proline was detected only in the seedlings of W₃, W₄ and W₅ treatments after February and was highest in the seedlings of W₅ treatment. The study suggests that severe water deficit adversely affect physiological and biochemical processes that resulted in reduced growth, nutrient uptake and biomass productivity in D. sissoo seedlings. Re-irrigation above W₃ level is recommended for this species.     

Mobility of Cytoplasmic, Membrane, and DNA-Binding Proteins in Escherichia coli

Protein mobility affects most cellular processes, such as the rates of enzymatic reactions, signal transduction, and assembly of macromolecular complexes. Despite such importance, little systematic information is available about protein diffusion inside bacterial cells. Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the bacterial cytoplasm, the plasma membrane, and in the nucleoid. Estimated diffusion coefficients of cytoplasmic and membrane proteins show steep dependence on the size and on the number of transmembrane helices, respectively. Protein diffusion in both compartments is thus apparently obstructed by a network of obstacles, creating the so-called molecular sieving effect. These obstructing networks themselves, however, appear to be dynamic and allow a slow and nearly size-independent movement of large proteins and complexes. The obtained dependencies of protein mobility on the molecular mass and the number of transmembrane helices can be used as a reference to predict diffusion rates of proteins in Escherichia coli. Mobility of DNA-binding proteins apparently mainly depends on their binding specificity, with FRAP recovery kinetics being slower for the highly specific TetR repressor than for the relatively nonspecific H-NS regulator.