Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Importance of fungi in the diet of Gammarus pulex and Asellus aquaticus

An important component of the interaction between macroinvertebrates and leaf litter in streams in the extent to which consumers can differentiate between undecomposed and decomposing leaves. The detritivores Gammarus pulex and Asellus aquaticus fed preferentially on conditioned rather on unconditioned leaf material. Growth in A. aquaticus was significantly reduced when unconditioned leaves were provided, but in G. pulex no significant effect of conditioning on growth was observed. The capacity of G. pulex to tolerate reductions in food quality seems to be a consequence of a compensatory system in which respiration rates change to compensate for reductions in food quality. In this way a constant growth rate is maintained. Increases in ingestion rates to compensate for low quality food were not observed.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Nitrogen losses in puddled soils as affected by timing of water deficit and nitrogen fertilization

Erratic rainfall in rainfed lowlands and inadequate water supply in irrigated lowlands can results in alternate soil drying and flooding during a rice (Oryza sativa L.) cropping period. Effects of alternate soil drying and flooding on N loss by nitrification-denitrification have been inconsistent in previous field research. To determine the effects of water deficit and urea timing on soil NO₃ and NH₄, floodwater NO₃, and N loss from added ¹⁵N-labeled urea, a field experiment was conducted for 2 yr on an Andaqueptic Haplaquoll in the Philippines. Water regimes were continuously flooded, not irrigated from 15 to 35 d after transplanting (DT), or not irrigated from 41 to 63 DT. The nitrogen treatments in factorial combination with water regimes were no applied N and 80 kg urea-N ha^–1, either applied half basally and half at 37 DT or half at 11 DT and half at 65 DT. Water deficit at 15 to 35 DT and 41 to 63 DT, compared with continuous soil flooding, significantly reduced extractable NH₄ in the top 30-cm soil layer and resulted in significant but small (<1.0 kg N ha^–1) soil NO₃ accumulations. Soil NO₃, which accumulated during the water deficit, rapidly disappeared after reflooding. Water deficit at 15 to 35 DT, unlike that at 41 to 63 DT, increased the gaseous loss of added urea N as determined from unrecovered ¹⁵N in ¹⁵N balances. The results indicate that application of urea to young rice in saturated or flooded soil results in large, rapid losses of N (mean = 35% of applied N), presumably by NH₃ volatilization. Subsequent soil drying and flooding during the vegetative growth phase can result in additional N loss (mean = 14% of applied N), presumably by nitrification-denitrification. This additional N loss due to soil drying and flooding decreases with increasing crop age, apparently because of increased competition by rice with soil microorganisms for NH₄ and NO₃.     

Models of patch dynamics in tropical forests

Recent interest in the ecology and evolution of metapopulations and conservation of fragmented populations has stimulated the development of models that combine patch and population dynamics in tropical forests. One approach uses matrix models that are actual metapopulation or multi-regional demographic models. Another approach uses computer simulations to model forest succession based on the behavior of individual trees. We review applications of both types of models and suggest new combined modelling approaches.     

Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures

Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations. Correspondence to: C. Nombela     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Sperm concentration influences fertilization and male pronuclear formation in vitro in pigs

To study the effect of sperm concentration on the results of pig in vitro fertilization (IVF), 313 oocytes recovered from oviducts of prepubertal gilts after induction of ovulation were used. After capacitation, the number of live spermatozoa in the fertilization dishes was adjusted to 3 x 10(5), 6 x 10(5) and 12 x 10(5) cell/ml. After 4 hours of co-culture in TCM-199, the oocytes were pippeted to remove cumulus cells and the excess spermatozoa around the zona pellucida, and were transferred to fresh TCM-199 for another 12 14 hours . The results showed that 6 x 10(5) spermatozoa/ml is the optimum concentration for this system; the percentage of fertilized ova (71.6%) was not different from the best (76.8%), that was obtained with the highest concentration, and the percentage of monospermy (62.3%) was not different from the best (68.1%), that was obtained with the lowest concentration. The percentage of spermatozoa that reached the pronuclear stage increased while sperm concentration was decreased. The percentage of spermatozoa at the decondensed stage was decreased when the sperm concentration increased.     

Three new HLA-G alleles and their linkage disequilibria with HLA-A

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A T), but conservative, (Thr Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A T) at the third base of codon 107 and (G A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.The nucleotide sequence data reported in this paper have been submitted to the GenBank and EMBL nucleotide sequence databases and have been assigned the following accession numbers: EMBL-X60983 (HLA-G*II), GenBank-M99048 (HLA-G*III), and GenBank-L07784 (HLA-G*IV).The contribution to this paper by P. Morales and A. Corell is equal, and the order of authorship is arbitrary. Correspondence to: A. Arnaiz-Villena.     

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

Summary Ubiquinone-10 (Q₁₀) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O₂ in the sparging gas). Whereas the cellular level of Q₁₀ decreased monotonically from 1.2 to 0.5 mol/g d.w., the total yield of Q₁₀ was maximal at 2.5 % O₂ and amounted to 350 nmol (0.3 mg) per L of culture.