The formation of triple-stranded DNA prevents spontaneous branch-migration

Branch-migration is a fundamental step in the process of DNA recombination that determines the location, and extent, of the exchange between the recombining duplexes. Four-way Holliday junctions assembled in vitro can migrate spontaneously in an uncatalysed reaction that mimics some of the aspects involved in branch-migration. Here, we have analysed the effects of a d(GA.TC)22 and a d(CA.TG)30 sequence on the rate of spontaneous branch-migration. Under most of the experimental conditions assayed, no significant effect was observed. However, the d(GA.TC)22 sequence induces a very strong arrest when branch-migration is performed at low pH, under conditions where the repeated sequence is forming an intramolecular [C(+)T(GA.TC)] triplex. A similar arrest is observed when the recombining duplexes contain intermolecular triplexes arising from the annealing of a d(GA.TC)22 duplex and a d(TC)22 oligonucleotide, indicating that the formation of triplex DNA constitutes a strong barrier for the progression of the Holliday junction. These results are discussed in the context of the possible contribution of triplex DNA to DNA recombination.     

Functional analysis of the p40 and p75 proteins from Lactobacillus casei BL23

The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria.     

Tubulin-colchicine complex inhibits microtubule elongation at both plus and minus ends

We studied the mechanism by which tubulin-colchicine complex (TC) inhibits microtubule polymerization in vitro by using the axoneme-directed polymerization system (Bergen, L. G., and Borisy, G. G. (1980) J. Cell Biol. 84, 141-150). With this system, the growth properties of each microtubule end can be determined from the direct visual analysis of changes in lengths of seeded microtubules. The rate of growth at both ends was inhibited equally by TC and the magnitude of the inhibition increased progressively with the molar ratio of TC to tubulin dimer (TC:T). At a TC:T ratio of approximately 0.12, all microtubule polymerization was inhibited at both ends. Therefore, substoichiometric poisoning of microtubule elongation is both a nonpolar and graded phenomenon. We determined the four association and dissociation rate constants in the presence and absence of TC and found that TC inhibits the overall growth of microtubules by reducing the association rate constants at both ends under conditions that do not alter the dissociation rate constants. Therefore, by an independent analytical method, we have confirmed Sternlicht and Ringel's hypothesis of TC action (Sternlicht, H., and Ringel, I. (1979) J. Biol. Chem. 254, 10540-10550), and have extended this hypothesis 1) by demonstrating that net growth of both ends are equally inhibited by TC, and 2) by determining which changes in the separate rate constants were responsible for the net inhibition.     

Kinetics of leaf nitrite reductase with Methyl Viologen and ferredoxin under controlled redox conditions.

The dexamethasone binding capacity of embryonal carcinoma cells and their differentiated derivatives was investigated. Manipulation of the embryonal carcinoma cell-culture conditions resulted in an unstable reversible expression of the glucocorticoid receptors. Stable expression of the receptors is observed when these cells are induced to differentiate. Cells grown under identical conditions were assayed for their ability to bind epidermal growth factor.     

Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions

The mycorrhizal relationships between pines and two edible species of Lactarius sect. Dapetes were investigated by optimizing the experimental conditions of mycelial growth and of mycorrhizal colonization of pine seedlings. In vitro mycelial growth of Lactarius deliciosus and L. sanguifluus was improved on a buffered medium containing glucose, amino acids, and vitamins. Two methods of mycorrhization of pines with Lactarius deliciosus were tested. The mycorrhizal colonization was rapid and intense under non-aseptic conditions with a low nutrient supply and without exogenous glucose. A positive influence of mycorrhizal colonization on Pinus sylvestris growth was subsequently observed. Under axenic conditions and with a high nutrient supply, mycorrhization was stimulated at 10 g/L of exogenous glucose, irrespective of the phosphorus concentration. At high phosphorus level (1 mM) and 0.1, 1.0, or 10.0 g/L glucose, growth of Pinus sylvestris was reduced by inoculation. Stability and development of Pinus spp./Lactarius deliciosus symbioses were assayed in a climatic chamber using containers filled with a synthetic substrate. Over a 2-year culture period, the root systems of the pine seedlings were heavily colonized by Lactarius deliciosus. One year following inoculation, Lactarius deliciosus fruit-body primordia appeared associated with Pinus sylvestris seedlings. Six months later, two mature basidiomata were obtained. This is the first report of soilless fruit-body formation of this edible mushroom.     

Glutamate Dehydrogenase Activity in Lemna perpusilla 6746: The Effects of Sucrose,Glucose and Fructose Addition to Growth Media

Lemna perpusilla Torr, strain 6746 clones were maintained under conditions of continuous illumination with various concentrations of sucrose, glucose or fructose added to the growth, medium. After two weeks of growth, plants were harvested and either assayed for total glutamate dehydrogenase activity or fractionated into one chloroplast-rich and one mitochondria-rich preparation and then assayed for glutamate dehydrogenase activity. In all assays for glutamate dehydrogenase it was necessary to add bovine serum albumin to the extraction medium in order to obtain sufficient enzyme activity for accurate and reproducible results. The presence of sucrose in the growth medium reduced glutamate dehydrogenase activity in all studies. When samples containing intact organelles were assayed, sucrose inhibition of activity appeared to occur primarily in the chloroplast fraction. Glucose, on the other hand, increased glutamate dehydrogenase activity in the chloroplast-rich fractions. Upon freeze-thawing differences between the various treatments were less obvious. The results from these studies indicate possible differences in sugar uptake and/or utilization in Lemna perpusilla.     

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.     

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.     

What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.     

Mutants of Escherichia coli with altered hydrogenase activity

Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2. They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Characterization of Amylolytic Enzymes, Having Both alpha-1,4 and alpha-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90 degrees C) and Pyrococcus furiosus (optimal growth temperature, 98 degrees C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140 degrees C. The addition of Ca also affected substrate binding, as evidenced by a decrease in K(m) for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the alpha-1,6 linkages in pullulan, alpha-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller alpha-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Physiology of Ex Planta Nitrogenase Activity in Rhizobium japonicum

Thirty-nine wild-type strains of Rhizobium japonicum have been studied for their ability to synthesize nitrogenase ex planta in defined liquid media under microaerobic conditions. Twenty-one produced more than trace amounts of acetylene reduction activity, but only a few of these yielded high activity. The oxygen response curves were similar for most of the nitrogenase-positive strains. The strains derepressible for activity had several phenotypic characteristics different from non-derepressible strains. These included slower growth and lower oxygen consumption under microaerobic conditions and lower extracellular polysaccharide production. Extracellular polysaccharide production during growth on gluconate in every nitrogenase-positive strain assayed was lower under both aerobic and microaerobic conditions than the non-derepressible strains. These phenotypic characteristics may be representative of a genotype of a subspecies of R. japonicum. These studies were done in part to enlarge the base number of strains available for studies on the physiology, biochemistry, and genetics of nitrogen fixation.     

Overexpression of <Emphasis Type="Italic">ScALDH21</Emphasis> gene in cotton improves drought tolerance and growth in greenhouse and field conditions

Aldehyde dehydrogenase (ALDH) is essential for scavenging redundant aldehydes when plants are exposed to stress. The aim of the present study was to validate the ectopic expression of the ScALDH21 gene, which is isolated from Syntrichia caninervis, an extremely drought-tolerant moss, to improve drought tolerance in cotton (Gossypium hirsutum L.). In our study, the ScALDH21-transformed cotton was identified via PCR, RT-PCR, and DNA gel blotting, and the growth and physiological characteristics related to drought tolerance were compared between the transgenic cotton (TC) and non-transgenic cotton (NT) grown in a greenhouse and in field conditions. The results indicated that TC accumulated approximately 11.8–304 % more proline than did NT under drought stress, and produced a lower concentration of lipid peroxidation-derived reactive aldehydes and had a higher peroxidase activity under oxidative stress. Moreover, TC showed reduced loss of the net photosynthetic rate compared with NT. Under field conditions, TC showed greater plant height, larger bolls, and greater cotton fiber yield than NT, but no significant difference in fiber quality between TC and NT following different water-withholding treatments. These results suggest that overexpression of ScALDH21 can greatly improve the drought tolerance of cotton without reduction in yield and fiber quality.     

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Denitrification enzymes of Bacillus stearothermophilus

Abstract A hybrid trpPO:lacO regulatory sequence was cloned upstream of a promoterless lacZ gene and recombined onto a Λ bacteriophage. Escherichia coli lysogens representing the four possible phenotypes for lacI and trpR were constructed and the synthesis of β -galactosidase was assayed under various growth conditions. The results illustrated that both control elements could be efficiently and independently regulated by the addition or omission of appropriate accessory molecules.     

Effect of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation.

Bacteroides fragilis cells were more sensitive to far-ultraviolet radiation under aerobic conditions than under anaerobic conditions. The percentage of pyrimidine dimers assayed after irradiation under both conditions was similar.     

DIEL VARIATIONS OF CARBOHYDRATES AND NEUTRAL LIPIDS IN NITROGEN‐SUFFICIENT AND NITROGEN‐STARVED CYCLOSTAT CULTURES OF ISOCHRYSIS SP.1

The goal of this study was to investigate the time response of two major carbon (C) reserves, respectively neutral lipids (NL) and total carbohydrate (TC), in the Haptophyte Isochrysis sp. growing in nitrogen (N)‐sufficient or N‐starved conditions and under light:dark (L:D) cycles. Experiments were carried out in a cyclostat culture system that allowed the following of the dynamics of the main cell compounds at both hourly and daily time scales. Under N‐sufficient conditions, the L:D cycles cause the population to be synchronized, with most of the cells dividing at the beginning of the dark period. The C‐specific growth rate was maximal around midday and negative during the dark period due to respiration processes. NL and TC both accumulated during the day and consumed during the night. We showed that NL and TC are highly dynamic compounds, as more than three quarters of NL and TC accumulated during the light period were consumed during the dark period. In contrast to NL, phospholipid and glycolipid to C ratios remained quite stable during the light/dark cycles. The major effect of N starvation on the NL and TC dynamics was to uncouple their diel variations from the L:D cycle, in two different ways depending on their respective role during short‐term acclimation. Whereas the TC per cell ratio increased rapidly to reach a stable value in response to N starvation, NL per cell continued to oscillate, but with a pattern out of phase with the L:D cycle.