Fig. 3: Rof is a conserved regulator of Rho in bacterial pathogens.

a Multiple sequence alignment of Rof. Representative Rof sequences retrieved from UniProt or Genbank are aligned. Secondary structures are shown above, mutated residues used for in vivo testing are marked below by asterisks. b Rof inhibits the growth of E. coli under osmotic stress. WT and mutant Rof proteins are overexpressed from pET22b vectors, with the empty pET22b plasmid used as vector control (pET). E. coli BL21 containing the indicated plasmids are grown overnight in LB supplemented with 0.6 M NaCl and 0.5 mM IPTG at 37 °C. The final OD is recorded and plotted. Data are presented as mean values ± SD from n = 6 biology replicates. c Rof inhibits Salmonella growth and colony formation in LB. Salmonella WT and Δrof mutant are transformed with either empty pZE12 vector (pEV), Rof complementation plasmid (pRof), or Rof overexpression plasmids (pL-Rof). pRof is constructed by cloning the rof gene with its own promoter into a promoterless pZE12 vector. pL-Rof contains the rof gene driven by a constitutive PLlacO promoter on pZE12. (Figure inset) few or no viable colony on LB plates is obtained after transformation with pL-Rof. After inoculating rare viable colonies into LB liquid medium containing 0.9 M NaCl, bacterial growth is monitored overtime. Data are presented as mean values ± SD from n = 3 biology replicates.