Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted ß-glucans, ß-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize ß-glucans and ß-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Small bait traps as accurate predictors of dipteran early colonizers in forensic studies.

Insect carrion communities vary among habitats and over time. Concerning the dipteran early colonizers of carrion, the use of small bait traps should be accurate because the odors emitted from meat baits should contain many of the volatile organic compounds emitted from the freshly dead mammals. In addition, this kind of trap is easy to replicate and set in position in a given habitat. In the present study, small bait preferences of early Diptera carrion colonizers were examined in an urban biotope. Specifically, three baits were compared (pork muscle, pork liver, and fish flavored cat food) in respect to the number of specimens and species captured and the presence or absence of oviposition at high and low environmental temperatures. A total of 2371 specimens were trapped, primarily belonging to three insect orders, Diptera, Coleoptera, and Hymenoptera. Diptera was the predominant order, with blowflies (Calliphoridae) being the most representative family, followed by filth flies (Muscidae). The pork muscle bait was responsible for the highest number of captures and the highest diversity. The community of Diptera collected with the most efficient bait, pork muscle, was compared with the carrion communities reported in the literature from the Iberian Peninsula. Similar taxonomic species composition was found regarding Calliphoridae species. A specimen from all species morphologically identified were also identified at a molecular level using the cytochrome c oxidase I (COI) barcode region, and the sequences were submitted to online databases.

Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics.

In recent years, forensic entomology has been applied in wildlife crimes, such as neglect cases, animal cruelty and illegal poaching. Likewise in human death investigations, in which insects can help to provide information about postmortem interval (PMI) and corpse transfer, entomology may be an important source of information in animal murder suspicion. The use of insects in forensic context relies primarily on its identification at the species level. To overcome some problems of morphological determination, molecular identification has gained relevance and has been applied frequently in forensic areas. Cytochrome c oxidase I (COI) gene was adopted in DNA barcoding approach. This methodology intends to unify the DNA-based identification using a specific region of mitochondrial DNA. COI sequences have been collected into the BOLD online database, allowing the molecular identification of sequences from unknown specimens. Nonetheless, to achieve a correct identification of an unknown sample, it is necessary that sequences from species under study exist, for comparison, in online databases. Due to the geographic differences, it is of huge importance to have samples from a certain species from its distribution range. In that sense, the aim of this research is to contribute to the potential and accuracy improvement of such databases in identification of species commonly found in wildlife carcasses. A portion of COI was sequenced from 95 specimens of seven species belonging to two families of Diptera (Calliphoridae and Muscidae) found in wildlife carcasses-baited traps in Serra da Estrela (Portugal). All specimens were identified at species level with a high specimen similarity and maximum identity percentage (through BOLD Systems and GenBank online databases, respectively). We also demonstrate the correct discrimination of all species through phylogenic and sequence divergence analyses proposed in DNA barcoding studies, reinforcing the suitability of this marker.