A01868

A C-terminal 2× repeat Flag-tag was inserted into C. jejuni fetA by FastCloning (35 (link)) using the constitutively expressing complementation vector pRRC_1651 as a template (Fig. S1K) and Q5 DNA polymerase. pRRC_1651 includes 93 C-terminal bp of fetP, 82 bp of the intergenic region, and the complete 1,404 bp of the fetA gene. C. jejuni strains ΔfetA and ΔfetABCDEF were then complemented with this construct following the method to generate ΔfetA^c, producing ΔfetA^2xFlag-FetA and ΔfetABCDEF^2xFlag-FetA. Successful insertion was confirmed by sequencing purified genomic DNA. Methods to confirm the proper functionality of the tagged FetA variant in the deletion strains are described in the Supplemental Methods.
To examine the expression of tagged FetA in C. jejuni, the variant-complemented strains were grown to mid-log-phase in 15 mL MH-TV, resuspended in fresh MH-TV supplemented with or without 10 µM DFO, and pelleted after 3 h of growth. The harvested cell pellets were analyzed by SDS-PAGE and probed by western blot with an anti-Flag antibody (Genscript A01868).

Membrane and cytosolic proteins were isolated following published protocols with modifications^{83 (link),84 (link)}. Briefly, the same dry weight of titan cells (CUX1331, CRK1^∆PESTP_GDP1-GPA1:FLAG) and typical cells (CUX1197, crk1∆ P_GDP1-GPA1:FLAG) were used for protein extraction. CUX1331 and CUX1197 were harvested and stored at −80 °C after culturing in titan cell inducing condition for 3 days. Samples were lyophilized and the same dry weight of lyophilized samples were subsequently pulverized with a ceramic mortar and a pestle and resuspended in 1.5 ml lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 µM PMSF and 1× EDTA-free protease inhibitor). Cells were centrifuged at 2000 × g for 10 min at 4 °C to remove non-lysed cells from total cells lysates. The supernatant was transferred to a new tube and separated into a soluble and pellet fraction by centrifugation at 25,000 × g for 60 min at 4 °C. The insoluble pellet was resuspended in 0.1 ml lysis buffer with 1% Triton X-100 to extract membrane proteins. 10 µl of soluble cytosolic proteins and 5 µl of membrane proteins were analyzed by western blotting with FLAG antibody (Genscript, A01868, 1:2000) and actin antibody (Genscript, A00702, 1:5000), marker of cytoplasm.