Expression of A. biwaensis HHJ01_10880 in A. muciniphila MucT

The gene encoding the putative A. biwaensis GH29 fucosidase HHJ01_10880 (clade 4) was introduced into A. muciniphila Muc^T using a modified mariner transposon (Tn) delivery system (pSAM_Akk) (67). The plasmid was re-engineered to insert genes within the inverted repeats of the Tn system and enable the expression of genes of interest in A. muciniphila under the control of the promoter of Amuc_1505 (RNA pol) promoter. The resulting plasmid (pE_Akk) contains a cat resistance cassette and the Amuc_1505 promoter flanked by Mariner transposase recognition sites.

The coding sequence of HHJ01_10880 was amplified by PCR from A. biwaensis Akk2750 (100% sequence match to CSUN-19) (38) with Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs) and incorporating a C-terminal HA epitope tag. The amplicon was inserted into SalI- and FseI-digested pE-Akk via In-Fusion Snap Assembly (Takara Bio). The correct sequence of pE_Akk_10880 was confirmed by Nanopore sequencing (Plasmidsaurus).

pE_Akk_10880 was introduced into E. coli S17-1 λpir strain for conjugative transfer into A. muciniphila Muc^T as outlined by Davey et al., with minor modifications (67). Briefly, the E. coli strain carrying pE_Akk_10880 and A. muciniphila Muc^T were co-incubated ~16 hours in a 10:1 under aerobic conditions on synthetic agar plates [3 mM KH₂PO₄, 3 mM Na₂PO₄, 5.6 mM NH₄Cl, 1 mM MgCl₂, 1 mM Na₂S·9H₂O, 47 mM NaHCO₃, 1 mM CaCl₂, 40 mM HCl, trace elements and vitamins, 0.2% GlcNAc, 0.2% glucose, 16 g l⁻¹ soy peptone, 4 g l⁻¹ threonine, 1.25% agar (68, 69)] with 0.5 g l⁻¹ thioglycolate added and lacking the added sugars (GlcNAc and glucose). After conjugation, cells were diluted and transferred into a synthetic medium supplemented with 20 μM ciprofloxacin and grown anaerobically for 48 hours. Bacterial cells were passaged twice by diluting 1:10 into fresh medium for 24 hours, before spreading onto synthetic agar plates containing an additional 0.25% mucin with ciprofloxacin and chloramphenicol (7 μg/mL) and incubated at 37°C until colonies were large enough to pick (~6 days). Four chloramphenicol-resistant colonies were picked and Tn insert locations mapped by arbitrary PCR.

Expression of HHJ01_10880 was confirmed via western blot (Fig. S1). Samples were separated on a 12% SDS-PAGE gel and subsequently blotted on nitrocellulose membranes (Bio-Rad, 0.22 µM) for 1 hour at 100V. Membrane blots were blocked with phosphate-buffered saline containing 0.2% Tween-20 (vol/vol, PBS-T) and 5% nonfat milk (wt/vol, 5% milk) for 1 hour. Each membrane was incubated with rat anti-HA primary antibody (Sigma 11867423001; clone 3F10, monoclonal from Roche, 1:5,000 in 5% milk) overnight at 4°C. Blots were washed 3× for 5 minutes in PBS-T at room temperature. Licor anti-rat-680 secondary antibody (LICOR IRDye 680LT Goat Anti-Rat IgG (H + L), Highly Cross-Adsorbed; LIC-925–68029) was added for 1 hour at room temperature (1:15,000). Blots were washed 3× for 5 minutes in PBS-T at room temperature and developed in the Odyssey Fc Imaging System (LI-COR Biosciences). All four mutants were used in preliminary comparative growth experiments with 2′-FL as described above; however, the clone that had the brightest band was named Akk-EH114 (MucT TnAmuc_2072::HHJ01_10880).