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Expression profiling of Cellvibrio japonicus using starch

ABSTRACT: Recent interest in rare α-diglucosides such as kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6) reflects developments in pharmaceutical, biotechnological, and culinary industries that value these sugars as prebiotics, carrier molecules, and low glycemic index sweeteners. There have been only a few enzymes capable of degrading these substrates characterized, largely due in part to difficulties in identifying potential targets based solely on bioinformatic predictions. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize rare α-diglucosides identified multiple, but thus far uncharacterized, mutations in each strain. In this report we analyzed the 36, 44, and 60 mutations that were in the kojibiose, nigerose, and isomaltose-adapted strains, respectively. The majority of mutations were unique to a specific adapted strain, which included indels that resulted a truncated protein, and single nucleotide variations within complete proteins. A single nucleotide variation in the C-terminus cyclomaltodextrin domain of the amy13E gene product (P606S) was observed in several adapted strains. RNAseq data identified amy13E as highly expressed in starch media, which suggested a key role for this gene in the metabolism of sugars with α-glycosidic bonds. Mutational analysis of amy13E found that this gene was essential for rare α-diglucoside metabolism and critical for the maintenance of adaptation phenotypes. Bioinformatic analysis coupled with biochemical assays using cell-free extracts indicated that the amy13E gene product is located in the periplasm and directly cleaves α-diglucosides into glucose. Our revised model of α-diglucoside degradation by C. japonicus is likely to be useful for making functional enzyme predictions in related bacteria.

ORGANISM(S): Cellvibrio japonicus

PROVIDER: GSE206866 | GEO | 2022/06/28

REPOSITORIES: GEO

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Project description:Background: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to an innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Results: Combined bioinformatic and transcriptomic analyses implicated three Glycoside Hydrolase Family 5 Subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Conclusions: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatics predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Project description:Transcriptome profiling of E. coli MG1655, E. coli EDL933, E. coli ECOR26, and E. coli Nissle 1917 grown on mouse cecal mucus as carbon source The carbon sources that support growth of pathogenic E. coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, pathogenic E. coli EDL933 primarily grows as dispersed single cells within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogen and commensal E. coli MG1655 for their mode of metabolism in vitro on a mixture of the sugars known to be present in cecal mucus and found that the two strains used the thirteen sugars in a similar order and co-metabolized as many as nine sugars at a time. We conducted a systematic mutational analysis of E. coli EDL933 and E. coli MG1655 with lesions in the pathways used for catabolism of thirteen mucus-derived sugars and five other compounds for which the corresponding gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both genetic backgrounds was fed to streptomycin-treated mice together with the respective wildtype parent strain and their colonization was monitored in fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive in E. coli EDL933, but not E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota. Keywords: growth condition: carbon source was mouse cecal mucus E. coli strains were grown on MOPS minimal medium containing 10 mg/ml of mucus or 0.2% glucose in 10 ml standing culture in testtubes and harvested at OD600 = 0.4.

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Expression profiling of Cellvibrio japonicus using starch

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