Microform-related community patterns of methane-cycling microbes in boreal Sphagnum bogs are site specific.

Vegetation and water table are important regulators of methane emission in peatlands. Microform variation encompasses these factors in small-scale topographic gradients of dry hummocks, intermediate lawns and wet hollows. We examined methane production and oxidization among microforms in four boreal bogs that showed more variation of vegetation within a bog with microform than between the bogs. Potential methane production was low and differed among bogs but not consistently with microform. Methane oxidation followed water table position with microform, showing higher rates closer to surface in lawns and hollows than in hummocks. Methanogen community, analysed by mcrA terminal restriction fragment length polymorphism and dominated by Methanoregulaceae or 'Methanoflorentaceae', varied strongly with bog. The extent of microform-related variation of methanogens depended on the bog. Methanotrophs identified as Methylocystis spp. in pmoA denaturing gradient gel electrophoresis similarly showed effect of bog, and microform patterns were stronger within individual bogs. Our results suggest that methane-cycling microbes in boreal Sphagnum bogs with seemingly uniform environmental conditions may show strong site-dependent variation. The bog-intrinsic factor may be related to carbon availability but contrary to expectations appears to be unrelated to current surface vegetation, calling attention to the origin of carbon substrates for microbes in bogs.

Chloroplasts assemble the major subunit FaeG of Escherichia coli F4 (K88) fimbriae to strand-swapped dimers.

F4 fimbriae encoded by the fae operon are the major colonization factors associated with porcine neonatal and postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 fimbriae are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytic degradation in the absence of a specific periplasmic chaperone. In order to test the possibility of producing FaeG in plants, FaeG expression was studied in transgenic tobacco plants. FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG to the chloroplast results in much higher yields than FaeG targeting to the endoplasmic reticulum or the apoplast. Two chloroplast-targeted FaeG variants were purified from tobacco plants and crystallized. The crystal structures show that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Furthermore, the structures reveal how FaeG combines the structural requirements of a major fimbrial subunit with its adhesive role by grafting an additional domain on its Ig-like core.

Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice.

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.

Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes.

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.