Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.     

Effect of Leaf Detachment on Chlorophyll Fluorescence during Chilling Experiments

The effect of leaf detachment on chlorophyll fluorescence was analyzed for Zea mays, Cucumis sativus, Phaseolus vulgaris, and Echinochloa crus-galli. Results clearly indicate that detachment hastens the decrease in chlorophyll fluorescence during the course of chilling experiments. For maize and bean, the activity of photosystem II of chloroplasts isolated from detached leaves is lower than that of chloroplasts isolated from attached leaves. There are also large differences in ionic loss between detached and attached leaves of barnyard grass which could correlate with changes in leaf water status. The detached leaves lost some 50% of their total ionic content. Finally, detachment alters the ranking of the species with regard to their chilling tolerance.     

Conservation genetics of the threatened horned grebe (<Emphasis Type="Italic">Podiceps auritus</Emphasis> L.) population of the Magdalen Islands,Québec

The horned grebe (Podiceps auritus) population of the Magdalen Islands in the St. Lawrence Gulf (Québec, Canada) has declined sharply over the last decades. It is the only breeding population of this species in eastern North America with nearest breeding populations being >2500 km apart in western North America and Europe, We used three types of genetic markers: mitochondrial (mt) DNA ND2 sequence, α-enolase intron sequence, and 25 amplified fragment length polymorphism loci (AFLPs) to quantify the genetic diversity within the Magdalen Island population and to assess its genetic distinctiveness relative to populations from western Canada (five sites) and Iceland (one site). The Magdalen Island population retained a comparable amount of genetic diversity to the average diversity observed across all populations in all three markers. Horned grebe mtDNA sequences formed a monophyletic group and nearly all haplotypes present in Québec were found elsewhere. In the ND2 fragment, populations partitioned into two groups corresponding to subspecies (Iceland versus North American sites) and more strongly in three groups according to geographic disjunctions (Iceland versus Québec versus western Canada). In contrast, there was no evidence of structure between sites in the α-enolase intron. In the AFLPs, Iceland showed the greatest level of differentiation, followed by the Québec and British Columbia populations. For conservation purposes, we suggest that the Magdalen Islands population should be recognized as a separate unit.     

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera)

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.     

Effects of muscle kinematics on surface EMG amplitude and frequency during fatiguing dynamic contractions

Potvin, J. R. Effects of muscle kinematics on surfaceEMG amplitude and frequency during fatiguing dynamic contractions. J. Appl. Physiol. 82(1): 144-151, 1997.Fifteen male subjects performed a repetitive elbowflexion/extension task with a 7-kg mass until exhaustion. Average jointangle, angular velocity, and biceps brachii surface electromyographic(EMG) amplitude (aEMG) and mean powerfrequency (MPF) were calculated with each consecutive 250-ms segment ofdata during the entire trial. Data were separated into concentric oreccentric phases and into seven 20°-ranges from 0 to 140° ofelbow flexion. A regression analysis was used to estimate the restedand fatigued aEMG and MPF values. aEMG values were expressed as apercentage of amplitudes from maximum voluntary contractions (MVC).Under rested dynamic conditions, the average concentric aEMG amplitudewas 10% MVC higher than average eccentric values. Rested MPF valueswere similar for concentric and eccentric phases, although valuesincreased ~20 Hz from the most extended to flexed joint angles.Fatigue resulted in an average increase in concentric and eccentricaEMG of 35 and 10% MVC, respectively. The largest concentric aEMGincreases (up to 58% MVC) were observed at higher joint velocities,whereas eccentric increases appeared to be related to decreases invelocity. Fatigue had a similar effect on MPF during both concentricand eccentric phases. Larger MPF decreases were observed at shortermuscle lengths such that values within each angle range were verysimilar by the end of the trial. It was hypothesized that this findingmay reflect a biological minimum in conduction velocity beforepropagation failure occurs.

     

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.     

Tree species richness affects litter production and decomposition rates in a tropical biodiversity experiment

We report data on leaf litter production and decomposition from a manipulative biodiversity experiment with trees in tropical Panama, which has been designed to explore the relationship between tree diversity and ecosystem functioning. A total of 24 plots (2025 m²) were established in 2001 using six native tree species, with 1‐, 3‐, and 6‐species mixtures. We estimated litter production during the dry season 2005 with litter traps; decomposition was assessed with a litter bag approach during the following wet season. Litter production during the course of the dry season was highly variable among the tree species. Tree diversity significantly affected litter production, and the majority of the intermediate diverse mixtures had higher litter yields than expected based on yields in monoculture. In contrast, high diverse mixtures did not show such overyielding in litter production. Litter decomposition rates were also highly species‐specific, and were related to various measures of litter quality (C/N, lignin/N, fibre content). We found no overall effect of litter diversity if the entire litter mixtures were analyzed, i.e. mixing species resulted in pure additive effects and observed decomposition rates were not different from expected rates. However, the individual species changed their decomposition pattern depending on the diversity of the litter mixture, i.e. there were species‐specific responses to mixing litter. The analysis of temporal C and N dynamics within litter mixtures gave only limited evidence for nutrient transfer among litters of different quality. At this early stage of our tree diversity experiment, there are no coherent and general effects of tree species richness on both litter production and decomposition. Within the scope of the biodiversity‐ecosystem functioning relationship, our results therefore highlight the process‐specific effects diversity may have. Additionally, species‐specific effects on ecosystem processes and their temporal dynamics are important, but such effects may change along the gradient of tree diversity.     

A Streptomyces chitosanase is active in transgenic tobacco

Summary Growth inhibition towards Rhizopus nigricans, Fusarium oxysporum f. sp. radicis-lycopersici, Verticillium albo-atrum and Pythium ultimum was observed in vitro using a purified chitosanase from an actinomycete, Streptomyces sp, strain N174. The corresponding gene, with its own signal peptide, was inserted into pBI121.7 shuttle vector to transform tobacco. Transgenic plants were analysed for chitosanase activity by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay. Two major and one minor active electrophoretic forms were detected in transgenic tobacco. Some chitosanases were recovered not only in leaf homogenates but also in leaf intercellular fluid extracts. One chitosanase electrophoretic form migrated very closely to the purified Streptomyces mature protein while the others corresponded to molecules of higher molecular mass. The N-terminus sequence was determined for one of the three chitosanase forms. It exhibited a different signal peptide cleavage site when compared to the mature chitosanase from Streptomyces. This is the first report on the expression of an active chitosanase gene with antimicrobial potential in plants.Abbreviations aa amino acid - CIP calf intestinal phosphatase - CM carboxymethyl - GUS ß-glucuronidase - IF intercellular fluid - MS Murashige and Skoog - PAGE polyacrylamide gel electrophoresis - PR pathogenesisrelated - PVDF polyvinylidene difluoride - SP signal peptide