D maltose monohydrate

ZnO NPs were purchased from Sumitomo (Tokyo, Japan) and dispersed in DW (5 mg/mL) for 30 min prior to experiments. Particle size and morphology were determined by SEM (FEIQUANTA 250 FEG, Hillsboro, OR, USA). XRD patterns for NPs were measured using an X-ray diffractometer (D2phaser, Bruker AXS Inc., Madison, WI, USA) with Ni-filtered CuKα radiation. Zeta potentials and hydrodynamic radii of NPs in aqueous suspension were measured with Zetasizer Nano System (Malvern Instruments, Worcestershire, UK). Materials used were as follow: acacia honey (Dongsuh Food Co., Ltd., Seoul, Republic of Korea), d-(+)-glucose (Sigma-Aldrich, St. Louis, MO, USA), d-(−)-fructose (Sigma-Aldrich), sucrose (Sigma-Aldrich), d-(+)-maltose monohydrate (Sigma-Aldrich).

The following compounds were purchased from Sigma-Aldrich: D-glucose ≥ 99.5%, D (−) fructose ≥ 99%, D (+) Turanos ≥ 98%, erlose ≥ 94%, isomaltotriose, D (+) melibiose ≥ 99,0%, D (+), raffinose pentahydrated ≥ 98.0%, palatinose hydrate ≥ 99%, sucrose ≥ 99.5%, D (+) maltose monohydrate min 98%, melezitose ≥ 99.0%, trehalose dihydrate (Certified Reference Materials), D-panose ≥ 88 97%, maltotriose, isomaltose 98%,ascorbic acid, gallic acid, 2,2-diphenyl-1 picrylhydrazyl (DPPH), Folin–Ciocalteu’s reagent, hexamethyldisilazane ≥ 99%, trifluoroacetic acid, hydroxylamine hydrochloride, acetic anhydride, pyridine, potassium hexacyanoferrate (II) trihydrate, and zinc acetate dihydrate. Methanol, sulfuric acid, ethanol, and acetonitrile were purchased from Merck (Darmstadt, Germany). Aluminum chloride, sodium carbonate, sodium hydroxide, and sodium nitrite were purchased from Merck (Darmstadt, Germany). All chemicals used were of analytical grade.

D-(+)-maltose monohydrate (Sigma-Aldrich) was used to stimulate growth from overnight stocks and as a model substrate for upregulating starch utilization. The maltodextrins used in this study were mixtures of maltodextrins with dextrose equivalent 16.5–19.5 (MD1) (Sigma-Aldrich) and maltodextrins with dextrose equivalent 4.0–7.0 (MD2) (Sigma-Aldrich). The soluble substrates potato starch, pullulan, amylopectin, amylose, dextran, and glycogen were purchased from Sigma-Aldrich (St. Louis, MO, USA), as well as the insoluble cornstarch. The cyclodextrin series (α-, β-, and γ-) were purchased from the American Maize Products Company (Stamford, CT, USA). Native potato starch was purchased from Bob’s Red Mill (Milwaukie, OR, USA). HIGH-MAIZE 260 cornstarch and VERSAFIBE 2470 were provided free of charge by Ingredion, Inc. (Westchester, IL, USA).

The strains of B. subtilis we used in this study are listed in S3 Table. We cultured B. subtilis strains at 37°C in lysogeny broth (LB) [1% tryptone (Bacto), 0.5% yeast extract (BBL), 0.5% sodium chloride (Sigma)] or on LB agar plates [1.5% Agar (Bacto)]. We maintained Streptomyces sp. Mg1 (PSK0558) as a spore stock in water at 4°C. Unless otherwise stated all co-cultures were grown on MYM [0.4% malt extract (Bacto), 0.4% yeast extract (BBL), 0.4% D-(+)-maltose monohydrate (Sigma)] with 2% agar (Bacto). We used chloramphenicol (5 μg/mL), kanamycin (5 μg/mL), MLS (1 μg/mL erythromycin, 25 μg/mL lincomycin), spectinomycin (100 μg/mL), and tetracycline (20 μg/mL) as needed. The primers we used in this study are listed in S4 Table. We used Escherichia coli DH5α or XL-1 blue for plasmid maintenance and manipulation. We prepared All B. subtilis genetic manipulations in either the 168 or PY79 strain background and then transduced them to NCIB3610 using SPP1 phage transduction as previously described [90 (link)].

CHO-K1 cells (American Type Culture Collection, Manassas, VA) adapted to maltose-only HyQ PF-CHO MPS (HyClone, Logan, UT)^{11 (link)} was further adapted into a DMEM/F12-based protein free chemically defined medium (PFCDM) supplemented with 10 g/L D-(+)-Maltose monohydrate (Sigma-Aldrich), 8 mM L-Glutamine (Sigma-Aldrich) and 0.1% Pluronic® F-68 (Life Technologies, Carlsbad, CA). An anti-Her2 monoclonal antibody-producing CHO-K1 cell line (SH87)^{13 (link)} was cultivated in PFCDM supplemented with 6 g/l D-(+)-Glucose (Sigma-Aldrich), 8 mM L-Glutamine (Sigma-Aldrich), 0.1% Pluronic F-68 (Life Technologies), and 600 µg/ml G418 disulfate salt (Sigma-Aldrich). Passaging of CHO-K1 and SH87 cells were performed every 3 to 4 days as reported previously^{11 (link)}.

Following column re-equilibration after a high-salt wash, MBP-DIX proteins were eluted with three column volumes of lysis buffer containing 10 mM D-(+)-maltose monohydrate (Sigma) applied to loose resin under gentle rotation for 30 min. After draining the first elution, one more column volume of elution buffer was mixed with the resin bed for 5 min to complete the elution. The combined eluate could be concentrated up to 15 mg/mL. Yields for soluble His₆-MBP-DIX range from 15 to 100 mg/L. For long-term storage at −80°C, the proteins were supplemented with 5% sucrose and subjected to flash-freezing with liquid nitrogen. For cleavage of His₆-MBP tag to trigger filament formation, 0.5 mg/mL TEV protease was mixed with His6-MBP-DIX at 1:35 v:v and incubated at 4°C overnight. Filaments were further purified using size-exclusion chromatography with a Superose 6 Increase 10/300 GL gel filtration column in lysis buffer containing 1 mM EDTA. For samples intended for electron microscopy, glycerol was omitted. Quantitative analysis of purified filaments by band intensity on stain-free SDS-PAGE gels indicates that the purified filament fractions contain ~10% uncleaved His₆-MBP-DIX. No electron density could be attributed to the MBP tag in the helical reconstruction.

The strains used in this work were obtained from the sources listed in Appendix␣Table S3. The three clinical C. difficile isolates (MS002, MS010, and MS011) were C. difficile NAAT (GeneXpert) positive via admission stool sample and toxin A (tcdA) and toxin B (tcdB) positive via in‐house research PCR. Each patient was diagnosed with and treated for CDI. Single‐use glycerol stocks were prepared as described previously (Clark et␣al, 2021 (link)). The media used in this work are anaerobic basal broth (ABB, Oxoid), clostridial reinforced medium (CRM, Difco), YP broth (Geva‐Zatorsky et␣al, 2015 (link)), and YBHI. YBHI broth recipe: BHI broth (Accumedia), 5 g/l yeast extract (BD Bacto), 1 g/l d‐cellobiose (Chem‐Impex), 1 g/l d‐maltose monohydrate (Sigma‐Aldrich), and 0.5 g/l l‐cysteine (Sigma‐Aldrich).