Author : Hannah Carney
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (14 download)
Book Synopsis Characterization of Carbohydrate Utilization in Human Gut Isolates by : Hannah Carney
Download or read book Characterization of Carbohydrate Utilization in Human Gut Isolates written by Hannah Carney and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Introduction: The human gut microbiome is an imperative ecosystem to understand as it impacts several biological processes in the body. It is known that short-chain fatty acids, such as butyrate, are likely the key bacterial metabolite that links the microbiome to health outcomes. Dietary resistant starch (RS) increases butyrate production through a cross-feeding mechanism between microorganisms able to utilize RS and butyrate-producers. Currently, there are only two known RS-degrading species: Ruminococcus bromii and Bifidobacterium adolescentis. However, recent strain-level differences in how R. bromii utilizes different types of carbohydrates as an energy source have been observed, challenging the notion that unique strains of these species contribute to microbial butyrate production in the same way. We hypothesized that there are additional uncharacterized bacteria residing in the human gut microbiome, besides B. adolescentis and R. bromii, that harbor the unique enzymatic system needed to utilize resistant starch in the colon. It was also hypothesized that different glucose transportation systems are present between different strains of R. bromii. Methods: In the present study, isolates obtained by an automated high-throughput isolation process from a previous dietary intervention study were screened by culture-based methods for ability to utilize soluble carbohydrates with varying structural complexities designed to mimic RS. DNA was extracted from isolates that displayed unique growth patterns on complex substrates and identified by 16S Sanger sequencing. Additionally, a series of screening steps were developed as an attempt to manually isolate RS-degrading isolates from the original RS-enriched fecal samples. Metagenomic data obtained from a previous dietary intervention study was also analyzed to identify and assemble R. bromii genomes with Megahit, CONCOCT, and Kraken2. PROKKA was used to annotate protein coding sequences in the de novo R. bromii genomes. The Transporter Classification Database and the NCBI blastp and tblastn databases were used to predict carbohydrate-active functional genes and transport proteins within the assembled genomes. Results: The isolates obtained through high-throughput isolation that demonstrated the ability to break down the most complex maltodextrin substrate were identified as Enterococcus spp. Despite successful manual isolation of colonies on anaerobic RS-enriched plates, these isolates did not demonstrate utilization of RS after conducting an iodine starch assay. Of the 31 potential Ruminococcus high-quality de novo assembled genome bins, 4 bins contained the following genes characteristic of known R. bromii strains: malP/malQ pair, melB, and a GH13 amylase or pullulanase. To further verify the successful isolation of possible R. bromii metagenome-assembled genomes (MAGs), two genome bins aligned very closely with a Ruminococcus and R. bromii reference genome, respectively. Although a few minor differences in sugar transport protein systems were observed between different strains, a glucose transporter was not identified in either isolate or the reference genomes used. Conclusion: Evidence of additional RS-degrading bacteria in the microbiome would enhance future research on the enzyme systems that have the capability to degrade resistant starch in the colon. The culture-based methods of isolation developed in this study could not confirm the hypothesis that novel RS-degrading species exist in the human gut microbiome. Additional culture-based methods need to be developed to achieve isolation and characterization of novel species. Additionally, there was no difference found in the genomes of the reference R. bromii strain L2- 63, known to be able to utilize RS, and the R. bromii strain L2-36, known to be able to use glucose. Despite minor differences in carbohydrate transporters among the two putative R. bromii MAGs pulled from the original metagenomic dataset, a glucose-specific transporter was not identified in either of these genomes. Therefore, a conclusion cannot be made about the ability of unique R. bromii strains to be able to utilize glucose. This study highlights the need for future studies focusing on the identification and characterization of glucose-specific ABC and PTS transport systems harbored by Firmicutes in the human gut microbiome.
