This experiment has been repeated at least three times with similar result. Duplicate biological replicates were used for each condition. Figure 2 Z. mobilis tolerance to different classes of pretreatment PF-6463922 solubility dmso inhibitors and Hfq. Z. mobilis strains were grown in RM (pH 5.0) overnight, 5-μL culture were then transferred into 250-μL RM media in the Bioscreen plate. The growth

differences of different strains were monitored by Bioscreen (Growth Curves USA, NJ) under anaerobic conditions in RM, pH 5.0 (A), RM with 1 g/L vanillin, pH 5.0 (B), 1 g/L furfural, pH 5.0 (C), and 1 g/L HMF, pH 5.0 (D). Hfq contributes to sodium and acetate ion tolerances: although the final cell density of hfq mutant AcRIM0347 is similar to that of AcR parental strain (Table 2; Fig. 2A), the growth rate of AcRIM0347 was reduced about one-fifth even without any inhibitor in the RM, which indicates hfq plays a central role in normal Z. mobilis physiology.

GS-9973 purchase Wild-type ZM4 that contained p42-0347 was able to grow in the presence of 195 mM sodium acetate and had a similar growth rate and final cell density to that of acetate tolerant strain AcR (Table 2; Fig. 1C). The wild-type ZM4 was unable to grow under this condition. The inactivation of the hfq gene in AcR decreased this acetate tolerant strain’s resistance to both sodium ion (sodium chloride) and acetate ion (ammonium acetate and potassium acetate) (Table 2; Fig. 1). hfq mutant AcRIM0347 was unable to grow in the presence of 195 mM ammonium acetate or potassium acetate (Table 2; Fig. 1D, E). Both the growth rate and final cell density of hfq mutant AcRIM0347 were reduced by at least a quarter in the presence of 195 mM sodium chloride, and about 60% in the presence of 195 mM sodium

acetate compared to that of the parental strain AcR (Table 2; Fig. 1B, C). The AcRIM0347 hfq mutation was complemented by the introduction of Nintedanib (BIBF 1120) an hfq-expressing plasmid (p42-0347) into the strain. The complemented mutant strain recovered at least half of the parental strains growth rate and 70% of its final cell density in the presence of 195 mM acetate ion (whether as sodium, ammonium or potassium acetate) (Table 2; Fig. 1). Hfq contributes to vanillin, furfural and HMF tolerances: AcRIM0347 growth rates were lower than that of ZM4 and AcR under all conditions tests, and except for growth in RM broth (Table 3; Fig. 2). AcRIM0347 also achieved lower final cell densities compared to ZM4 and AcR (Table 3; Fig. 2). When AcRIM0347 was provided functional Z. mobilis Hfq via p42-0347, growth rates under all conditions were largely unchanged (Table 3). However, shorter lag phases were observed for AcRIM0347 (p42-0347) grown with vanillin, furfural or HMF and increases in final cell densities were also observed under these conditions (Table 3; Fig. 2).