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. 2017 Feb 21;114(10):E1958–E1967. doi: 10.1073/pnas.1615056114

Fig. 7.

Fig. 7.

No detectable leak of xylitol across the cytoplasmic membrane when cells produce Tat translocases harboring TatB suppressors. (A) Overnight cultures of E. coli strain BW25113 ∆glpF ∆tatABC harboring pBAD24 encoding TatA and TatC-his along with wild type TatB or each of the L9Q, L10P, F13Y, or I36N point substitutions, were subcultured at 1:100 dilution into fresh LB medium containing ampicillin, which was supplemented after 120 min with 0.2% of glucose or arabinose, as indicated. Growth of the strains was followed for a further 6.5 h. Error bars represent SD, n = 3 (biological replicates). (B) The same strain and plasmid combinations as in A, alongside BW25113 ∆glpF harboring pBAD22SecY(∆plug)EG, were subcultured and supplemented with 0.2% of arabinose as described in A and grown for an additional 3 h, after which spheroplasts were prepared and incubated in the presence of xylitol. (C) An aliquot of each sample producing plasmid-encoded Tat proteins was analyzed by SDS/PAGE and Western blotting to confirm expression of TatA, TatB, and TatC-His.