Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web

Generalist predators have the capacity to restrict pest population growth, especially early in the season before densities increase. However, their polyphagous feeding habits sometimes translate into reduced pest consumption when they target alternative prey. An order-specific monoclonal antibody was developed to examine the strength of trophic connections between Diptera, a major category of non-pest prey, and linyphiid spiders in alfalfa. We report the development and characterization of a monoclonal antibody with order-level specificity to Diptera. This antibody elicited strong absorbance to 22 Diptera from 13 families, no false-positive reactivity to non-dipteran invertebrates, and antigen detection periods following prey consumption that were comparable between spiders. Over 900 field-collected females of the linyphiid spiders Erigone autumnalis and Bathyphantes pallidus were screened for Diptera antigen. Significantly more B. pallidus screened positive for Diptera (40%) compared to E. autumnalis (16%), indicating differential reliance on these prey. In parallel with the collection of spiders for gut-content analysis, prey availability was estimated at web sites. The two spiders exhibited different feeding responses to prey availability. Consumption of Diptera by B. pallidus was strongly correlated with Diptera abundance whilst the availability of other potential prey did not influence predation rates. Conversely, E. autumnalis did not prey upon Diptera in proportion to availability, but increased Collembola activity-density reduced dipteran consumption. Integration of molecular gut-content analysis with precise sampling of prey demonstrated how two closely related linyphiid spiders exhibit different feeding responses to the availability of prey under natural field conditions. Elucidating the feeding preferences of natural enemies is critical to effective incorporation of biological control by generalist predators in the management of agricultural pests.     

Exclosure fences around nests of imperiled Florida Grasshopper Sparrows reduce rates of predation by mammals

Increasing nest survival by excluding predators is a goal of many bird conservation programs. However, new exclosure projects should be carefully evaluated to assess the potential risks of disturbance. We tested the effectiveness of predator exclosure fences (hereafter, fences) for nests of critically endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) at a dry prairie site (Three Lakes; 2015–2018) and a pasture site (the Ranch; 2015–2016) in Osceola County, Florida, USA. We installed fences at nests an average of 8 days after the start of incubation, and nest abandonment after fence installation was rare (2 of 149 installations). Predation was the leading cause of failure for unfenced nests at both sites (48–73%). At Three Lakes, nest cameras revealed that mammals and snakes were responsible for 61.5% and 38.5% of predation events, respectively, at unfenced nests. Fences reduced the daily probability of predation (0.016 for fenced nests vs. 0.074 for unfenced nests). The probability that a fenced nest would survive from discovery to fledging was more than double that of unfenced nests (60.4% vs. 27.7%). However, we found no difference in daily nest survival at the Ranch between the year before nests were fenced (2015; 0.874) and the year when all but one nest were fenced (2016; 0.867) because red imported fire ants (Solenopsis invicta) were responsible for 86% of predation events at fenced nests at the Ranch. The use of cameras at fenced nests revealed that site‐specific differences in nest predators explained variation in fence efficiency between sites. Our fence design may be useful for other species of grassland birds, but site‐specific predator communities and species‐specific response of target bird species to fences should be assessed before installing fences at other sites.     

Sulfide induces phosphate release from polyphosphate in cultures of a marine Beggiatoa strain

Sulfur bacteria such as Beggiatoa or Thiomargarita have a particularly high capacity for storage because of their large size. In addition to sulfur and nitrate, these bacteria also store phosphorus in the form of polyphosphate. Thiomargarita namibiensis has been shown to release phosphate from internally stored polyphosphate in pulses creating steep peaks of phosphate in the sediment and thereby inducing the precipitation of phosphorus-rich minerals. Large sulfur bacteria populate sediments at the sites of recent phosphorite formation and are found as fossils in ancient phosphorite deposits. Therefore, it can be assumed that this physiology contributes to the removal of bioavailable phosphorus from the marine system and thus is important for the global phosphorus cycle. We investigated under defined laboratory conditions which parameters stimulate the decomposition of polyphosphate and the release of phosphate in a marine Beggiatoa strain. Initially, we tested phosphate release in response to anoxia and high concentrations of acetate, because acetate is described as the relevant stimulus for phosphate release in activated sludge. To our surprise, the Beggiatoa strain did not release phosphate in response to this treatment. Instead, we could clearly show that increasing sulfide concentrations and anoxia resulted in a decomposition of polyphosphate. This physiological reaction is a yet unknown mode of bacterial polyphosphate usage and provides a new explanation for high phosphate concentrations in sulfidic marine sediments.     

Effect of alkylresorcin on biological membranes during activation of lipid peroxidation

The effect of alkyl resorcin isolated from the cells of Azotobacter chroococcum and of its structural analog devoid of the alkyl chain (resorcin) on liver microsomes and brain synaptosomes of the rat as well as on rabbit skeletal muscle sarcoplasmic reticulum fragments during activation of lipid peroxidation was studied. Alkyl resorcin was shown to produce a much more potent antioxidant effect as compared with resorcin, since it inhibited lipid peroxidation in all the three types of membranes under study at much lower concentrations. Both alkyl resorcin and resorcin which inhibit lipid peroxidation prevented lipid peroxidation-induced structural-functional damages of synaptosomal and sarcoplasmic reticulum fragment membranes. Unlike resorcin, alkyl resorcin exerted an additional effect on brain synaptosomal membranes which consisted in the stabilization of barrier functions of membranes during incomplete inhibition of lipid peroxidation. The cumulative data suggest that stabilization necessitates the presence of both resorcin radical and alkyl chain in the alkyl resorcin molecule.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.