Six CO₂ fixation pathways are known to operate in photoautotrophic and chemoautotrophic microorganisms. Here, we describe chemolithoautotrophic growth of the sulphate-reducing bacterium Desulfovibrio desulfuricans (strain G11) with hydrogen and sulphate as energy substrates. Genomic, transcriptomic, proteomic and metabolomic analyses reveal that D. desulfuricans assimilates CO₂ via the reductive glycine pathway, a seventh CO₂ fixation pathway. In this pathway, CO₂ is first reduced to formate, which is reduced and condensed with a second CO₂ to generate glycine. Glycine is further reduced in D. desulfuricans by glycine reductase to acetyl-P, and then to acetyl-CoA, which is condensed with another CO₂ to form pyruvate. Ammonia is involved in the operation of the pathway, which is reflected in the dependence of the autotrophic growth rate on the ammonia concentration. Our study demonstrates microbial autotrophic growth fully supported by this highly ATP-efficient CO₂ fixation pathway.

Background: The plant-pathogenic fungus Fusarium oxysporum f.sp.lycopersici (Fol) has accessory, lineage-specific (LS) chromosomes that can be transferred horizontally between strains. A single LS chromosome in the Fol4287 reference strain harbors all known Fol effector genes. Transfer of this pathogenicity chromosome confers virulence to a previously non-pathogenic recipient strain. We hypothesize that expression and evolution of effector genes is influenced by their genomic context. Results: To gain a better understanding of the genomic context of the effector genes, we manually curated the annotated genes on the pathogenicity chromosome and identified and classified transposable elements. Both retro- and DNA transposons are present with no particular overrepresented class. Retrotransposons appear evenly distributed over the chromosome, while DNA transposons tend to concentrate in large chromosomal subregions. In general, genes on the pathogenicity chromosome are dispersed within the repeat landscape. Effector genes are present within subregions enriched for DNA transposons. A miniature Impala (mimp) is always present in their promoters. Although promoter deletion studies of two effector gene loci did not reveal a direct function of the mimp for gene expression, we were able to use proximity to a mimp as a criterion to identify new effector gene candidates. Through xylem sap proteomics we confirmed that several of these candidates encode proteins secreted during plant infection. Conclusions: Effector genes in Fol reside in characteristic subregions on a pathogenicity chromosome. Their genomic context allowed us to develop a method for the successful identification of novel effector genes. Since our approach is not based on effector gene similarity, but on unique genomic features, it can easily be extended to identify effector genes in Fo strains with different host specificities.

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [¹³C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [¹³C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.

Background: Peptide spectrum matching (PSM) is the standard method in shotgun proteomics data analysis. It relies on the availability of an accurate and complete sample proteome that is used to make interpretation of the spectra feasible. Although this procedure has proven to be effective in many proteomics studies, the approach has limitations when applied on complex samples of microbial communities, such as those found in the human intestinal tract. Metagenome studies have indicated that the human intestinal microbiome contains over 100 times more genes than the human genome and it has been estimated that this ecosystem contains over 5000 bacterial species. The genomes of the vast majority of these species have not yet been sequenced and hence their proteomes remain unknown. To enable data analysis of shotgun proteomics data using PSM, and circumvent the lack of a defined matched metaproteome, an iterative workflow was developed that is based on a synthetic metaproteome and the developing metagenomic databases that are both representative for but not necessarily originating from the sample of interest. Results: Two human fecal samples for which metagenomic data had been collected, were analyzed for their metaproteome using liquid chromatography-mass spectrometry and used to benchmark the developed iterative workflow to other methods. The results show that the developed method is able to detect over 3,000 peptides per fecal sample from the spectral data by circumventing the lack of a defined proteome without naive translation of matched metagenomes and cross-species peptide identification. Conclusions: The developed iterative workflow achieved an approximate two-fold increase in the amount of identified spectra at a false discovery rate of 1% and can be applied in metaproteomic studies of the human intestinal tract or other complex ecosystems.

Upon ingestion, nanoparticles can interact with the intestinal epithelial barrier potentially resulting in systemic uptake of nanoparticles. Nanoparticle properties have been described to influence the protein corona formation and subsequent cellular adhesion, uptake and transport. Here, we aimed to study the effects of nanoparticle size and surface chemistry on the protein corona formation and subsequent cellular adhesion, uptake and transport. Caco-2 intestinal cells, were exposed to negatively charged polystyrene nanoparticles (PSNPs) (50 and 200 nm), functionalized with sulfone or carboxyl groups, at nine nominal concentrations (15–250 μg/ml) for 10 up to 120 min. The protein coronas were analysed by LC–MS/MS. Subtle differences in the protein composition of the two PSNPs with different surface chemistry were noted. High-content imaging analysis demonstrated that sulfone PSNPs were associated with the cells to a significantly higher extent than the other PSNPs. The apparent cellular adhesion and uptake of 200 nm PSNPs was not significantly increased compared to 50 nm PSNPs with the same surface charge and chemistry. Surface chemistry outweighs the impact of size on the observed PSNP cellular associations. Also transport of the sulfone PSNPs through the monolayer of cells was significantly higher than that of carboxyl PSNPs. The results suggest that the composition of the protein corona and the PSNP surface chemistry influences cellular adhesion, uptake and monolayer transport, which might be predictive of the intestinal transport potency of NPs.

The nutritional value of insect protein is evaluated not only in amino acid composition, but also in protein digestibility. The general amino acid composition of Tenebrio molitor has been reported before, but limited knowledge is available on its digestibility. The objective of this study was to investigate in vitro protein digestibility of whole T. molitor larvae, a water-soluble fraction (supernatant) and water-insoluble fractions (pellet and residue), and to identify which proteins were present in the fractions studied. The digestibility of the supernatant fraction (~80 %) was much higher than that of pellet (~50 %) and residue (~24 %) after in vitro gastroduodenal digestion as was determined using the o-phthaldialdehyde (OPA) method. More proteins were digested after pepsin/pancreatin digestion than after only pepsin digestion. The most abundant proteins in the supernatant were hemolymph protein (~12 kDa), alpha-amylase (~50 kDa, a putative allergen), and muscle proteins (e.g. actin 30–50 kDa) in the pellet fraction as determined from LC–MS/MS and SDS-PAGE. In conclusion, the proteins in the soluble fraction that contained hemolymph proteins were more easily digestible than the insoluble, muscle protein-containing fractions.