Fig. 2: PUL33 is necessary for sialylated HMO utilization in B. dorei. | Nature Communications

Fig. 2: PUL33 is necessary for sialylated HMO utilization in B. dorei.

From: CRISPR-Cas-based identification of a sialylated human milk oligosaccharides utilization cluster in the infant gut commensal Bacteroides dorei

Fig. 2: PUL33 is necessary for sialylated HMO utilization in B. dorei.The alternative text for this image may have been generated using AI.

A Overview of the CRISPR-cas12-based genetic manipulation system, including the transformation of E. coli and conjugation to the recipient Bacteroides. Figure cartoon was created with BioRender.com [https://www.biorender.com/]. B PUL33 gene structure with GenBank locus tags, highlighting the regions of homology (LF: left flank, pink; RF: right flank, green), with colors matching to the plasmid in (A) and primer locations (P1–P4). The genes that are nonfunctional in the mutant are shaded in orange. SusC SusC-like TonB-dependent transporter, SusD SusD-like cell-surface glycan-binding protein, GH glycoside hydrolase, CE carbohydrate esterase, Est Sialate O-acetylesterase, Unk unknown function. C PCR results from wild-type (WT) colonies, representing failed Cas12a inductions in the attempt to create a mutant and a single mutant (KO) colony, with shorter PCR product (lacking the removed genes). D Growth curve plots of B. dorei, B. dorei Δ5k-PUL33 and B. dorei Δ5k-PUL33::p_compFull. Growth curves were measured in minimal media supplemented with sialylated HMOs (3’-SL, 6’-SL; dark and light pink, respectively), lactose (positive control; green) or no carbon media (negative control; gray), 0.5% weight/volume. Thick lines represent the average of two biological replicates, each consisting of three technical replicates, which are represented by thin lines (n = 6). p-values were calculated using a paired two-sided t-test at 24 h. E Structure overview of the plasmid p_compFull. Source data are provided as a Source Data file.

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