Bacteriophage-resistance mechanisms examined by transfection of Lactococcus lactis subsp. lactis 4513-5 mediated by high voltage electroporation

Summary Phage adsorption tests and transfection by electroporation were carried out to decide whether phage-resistance in Lactococcus lactis subsp. lactis strain 4513-5 is based on intracellular or extracellular mechanisms. Using high voltage (12.5 kV/cm) electroporation, untreated phage DNA was introduced into phage-sensitive and phage-resistant cells. Since phages showed low adsorption frequencies on resistant bacteria, resistance is localized in the cell wall preventing phage DNA from entering the cell. This is the only mechanism responsible for the resistance of L. lactis subsp. lactis 4513-5 against its homologous phage P4513-K12 and non-homologous phages P05M-13 and P05M-47, but not against phage P530-7 and phage P530-12. In the case of the latter two phage strains, intracellular resistance mechanisms are involved and discussed.     

Development and adaptation of a multiprobe biosensor for the use in a semi-automated device for the detection of toxic algae

Worldwide monitoring programs have been launched for the observation of phytoplankton composition and especially for harmful and toxic microalgae. Several molecular methods are currently used for the identification of phytoplankton but usually require transportation of samples to specialised laboratories. For the purpose of the monitoring of toxic algae, a multiprobe chip and a semi-automated rRNA biosensor for the in-situ detection of toxic algae were developed. Different materials for the electrodes and the carrier material were tested using single-electrode sensors and sandwich hybridisation that is based on species-specific rRNA probes. Phytoplankton communities consist of different species and therefore a biosensor consisting of a multiprobe chip with an array of 16 gold electrodes for the simultaneous detection of up to 14 target species was developed. The detection of the toxic algae is based on a sandwich hybridisation and an electrochemical detection method.     

Preparative use of isolated CYP102 monooxygenases -- a critical appraisal

Isolated P450 monooxygenases have for long been neglected catalysts in enzyme technology. This is surprising as they display a remarkable substrate specificity catalyzing reactions, which represent a challenge for classic organic chemistry. On the other hand, many P450 monooxygenases are membrane bound, depend on rather complicated electron transfer systems and require expensive cofactors such as NAD(P)H. Their activities are low, and stability leaves much to be desired. The use of bacterial P450 monooxygenases from CYP102 family allows overcoming some of these handicaps. They are soluble and their turnovers are high, presumably because their N-terminal heme monooxygenase and their C-terminal diflavin reductase domain are covalently linked. In recent years, protein engineering approaches have been successfully used to turn CYP102 monooxgenases into powerful biocatalysts.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Macroarthropod species richness and conservation priorities in Stratiotes aloides (L.) lakes

Species with narrow ranges and specialised traits are most at risk, and the extinction wave is further enhanced by coextinctions. We studied the conservation value and indicator potential of Stratiotes aloides, an aquatic macrophyte that has declined considerably in Europe. Our purpose was to determine whether S. aloides could be used as an indicator of a valuable habitat in terms of macroarthropod diversity and species richness. The potential occurrence of an internationally endangered Stratiotes-habitat specialist, the dragonfly Aeshna viridis, can increase the conservation value of plant colonies. S. aloides beds harboured diverse macroarthropod fauna often containing species of conservation concern, including A. viridis. Stratiotes is a potential indicator of a valuable habitat, and its indicator value is enhanced by the easy identification of the species. However, its use as an indicator of a defined macroarthropod community is limited because no particular community type is connected to it. We suggest that protecting Stratiotes simultaneously conserves valuable arthropod fauna, including A. viridis.     

Rethinking the common garden in invasion research

In common garden experiments, a number of genotypes are raised in a common environment in order to quantify the genetic component of phenotypic variation. Common gardens are thus ideally suited for disentangling how genetic and environmental factors contribute to the success of invasive species in their new non-native range. Although common garden experiments are increasingly employed in the study of invasive species, there has been little discussion about how these experiments should be designed for greatest utility. We argue that this has delayed progress in developing a general theory of invasion biology. We suggest a minimum optimal design (MOD) for common garden studies that target the ecological and evolutionary processes leading to phenotypic differentiation between native and invasive ranges. This involves four elements: (A) multiple, strategically sited garden locations, involving at the very least four gardens (2 in the native range and 2 in the invaded range); (B) careful consideration of the genetic design of the experiment; (C) standardization of experimental protocols across all gardens; and (D) care to ensure the biosafety of the experiment. Our understanding of the evolutionary ecology of biological invasions will be greatly enhanced by common garden studies, if and only if they are designed in a more systematic fashion, incorporating at the very least the MOD suggested here.