Construction of in-frame aroA deletion mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by using a new temperature-sensitive plasmid

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30 degrees C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.     

Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase

Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.     

Comparison of model selection technique performance in predicting the spread of newly invasive species: a case study with <Emphasis Type="Italic">Batrachochytrium salamandrivorans</Emphasis>

Species distribution models (SDMs) increasingly have been used to anticipate the spread of invasive species. However, these models are powerful conservation tools only if they are biologically relevant, and thus validation of these models is essential. Here, we evaluate four model selection frameworks for their ability to identify a best fit model of spread under low data conditions early in an invasion, specifically testing the efficacy of methods that utilize absence data in addition to presence data in evaluating models. We test this question using a simulation where we generated data with varying confidence in the accuracy of the absence data, as absences in early invasions may become presences in the future, and increasing quantity of observation data to test the models. We create these simulations based on a real-world example of a newly emergent, invasive fungal pathogen, Batrachochytrium salamandrivorans (Bsal). The simulation demonstrated that AIC and Likelihood consistently outperform both Kappa and AUC in selecting the true model as the best model when data are limited and absence data are low quality, with AIC providing the most conservative results due to penalties for overparameterization. With these results, we then used these techniques to compare five candidate models for predicting the spread of Bsal. Consistent with the simulation, the best fit model of the candidate models for Bsal was inconsistent across the four metrics. However, AIC, which performed best in the simulation study, suggested that the spread of Bsal into Western Europe was best predicted by a combination of bioclimatic suitability, salamander richness, and number of salamander imports. Our results highlight the difficulty in evaluating predictive models when data are limited and of low quality, as with a newly invasive species, but show that these challenges can be partially addressed with the appropriate model selection approach. Use of this approach is critical as SDMs of invasive species are often used to inform conservation policy and efforts before the invasion spreads, when limited data are available.     

Inhibition of Germination in Kochia indica

Seeds of Kochia indica Wight germinate rapidly in shallow water,but their germination is retarded on moist filter-paper. Theretardation is traced to a surface-active, saponin-like inhibitor,which is readily leached away in water and is adsorbed by charcoalor soil. Excised embryos may also remain dormant on filter-paper,but if rinsed in water quickly become active. Inhibition isfavoured by higher temperature (30°C. as against 20°or less), especially in an atmosphere of oxygen, although onceactive the embryos grow rapidly in such conditions. When theoxygen concentration is reduced to 5 per cent., germinationand growth are markedly retarded, but 5 per cent. CO₂ has littleor no retarding effect.