Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47-1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx-positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL-positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.     

Increased xerotolerance of Saccharomyces cerevisiae during an osmotic pressure ramp over several generations

Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; a_w) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing-to-destruction) at the rate of 1.5 MPa day^-1 from 1.38 to 58.5 MPa (0.990–0.635 a_w). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 a_w) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 a_w). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 a_w) showed a greater cultivability than glycerol-shocked cells after a subsequent shock at 200 MPa (0.234 a_w) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38–10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.     

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.     

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-gamma secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.     

Comparative multilocus phylogeography of two Palaearctic spruce bark beetles: influence of contrasting ecological strategies on genetic variation

While phylogeographic patterns of organisms are often interpreted through past environmental disturbances, mediated by climate changes, and geographic barriers, they may also be strongly influenced by species‐specific traits. To investigate the impact of such traits, we focused on two Eurasian spruce bark beetles that share a similar geographic distribution, but differ in their ecology and reproduction. Ips typographus is an aggressive tree‐killing species characterized by strong dispersal, whereas Dendroctonus micans is a discrete inbreeding species (sib mating is the rule), parasite of living trees and a poor disperser. We compared genetic variation between the two species over both beetles’ entire range in Eurasia with five independent gene fragments, to evaluate whether their intrinsic differences could have an influence over their phylogeographic patterns. We highlighted widely divergent patterns of genetic variation for the two species and argue that the difference is indeed largely compatible with their contrasting dispersal strategies and modes of reproduction. In addition, genetic structure in I. typographus divides European populations in a northern and a southern group, as was previously observed for its host plant, and suggests past allopatric divergence. A long divergence time was estimated between East Asian and other populations of both species, indicating their long‐standing presence in Eurasia, prior to the last glacial maximum. Finally, the strong population structure observed in D. micans for the mitochondrial locus provides insights into the recent colonization history of this species, from its native European range to regions where it was recently introduced.     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

Factors affecting pectate lyase activity during membrane filtration

Summary This paper presents problems encountered during ultrafiltration of pectate lyase (EC 4.2.2.2) of Erwinia chrysanthemi. Membrane adsorption was related to the chemical nature and charge of the membrane polymer. Simple pre-treatment such as coating with a lysine solution greatly improved recovery efficiency.     

Sphagnum physiological responses to elevated temperature,nitrogen, CO2 and low moisture in laboratory and in situ microhabitats: a review

Aquatic Ecology - Sphagnum mosses are considered peatland engineers because of their ability to create conditions inducing carbon accumulation. Here, we report on a review of the effects of four...     

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subspp. atroseptica

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpson's index of diversity (Simpson's D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0.46 (RFLP), 0. 39 (ERIC), 0.83 (phage typing), 0.82 (RAPD) and 0.26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.