Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications.

Project description:Human saliva has been commonly used as protein source in in vitro microbiological and biological assays to mimic the protein pellicle formation, termed acquired salivary pellicle, that precedes microbial and cell adhesion on surfaces exposed to the oral environment. However, saliva requires previous processing to remove food debris, microorganisms, and other molecules prior its use in microbiological and biological in vitro assays. For this purpose, 0.22 μm filtration, 0.45 μm filtration, and pasteurization methods have been commonly used, but the effect of these processing methods on the proteomic profile of saliva has not been tested experimentally. Stimulated human saliva was collected from 8 healthy volunteers and submitted to the following processes: non-processing (control), 0.22 μm filtration, 0.45 μm filtration, and pasteurization. The proteomic profile of non-processed saliva was compared with 0.22 μm filtered-, 0.45 μm filtered-, and pasteurized-saliva by liquid chromatography-mass spectrometry. The effect of processed saliva in microbial adhesion was tested using bacterial and fungus species, and in biological cell behavior using HaCaT immortalized human keratinocytes. Two hundred seventy-eight proteins were identified in non-processed saliva, 54 proteins (≈19%) were exclusive. Saliva processing reduced identified proteins to 222 (≈80%) for the 0.22 μm filtered saliva, 219 (≈79%) for the 0.45 μm filtered saliva, and 201 (≈72%) for the pasteurized saliva, compared to non-processed saliva. Although there were slight differences in the protein composition, the proteomic profile showed similar molecular functions and biological processes. The different saliva processing methods did not alter microbial adhesion (ANOVA, p>0.05). Interestingly, pasteurized saliva reduced keratinocytes cell viability. Saliva processing methods tested reduced the proteomic profile diversity of saliva, but maintained similar molecular functions and biological processes mediated by remaining proteins, not interfering on microbial adhesion and cell viability, except for pasteurization, which reduced cell viability.

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