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2 Fig. 1. Mechanisms by which Hfq might facilitate srna-mrna basepairing. Hfq (green ring) may promote RNA unfolding or may increase the local concentrations of the srna (red) and its mrna target (blue) 23). I 1 RNA Hfq β I 2 Hfq hfq hfq hfq hfq

3 Fig. 2. Phenotypes and factors controlled by the RNA chaperone Hfq. Table 1. Susceptibility of E. coli strains to toxic compounds MIC ( g/ml) Strain CP NOV ACR CV R6G BENZ OXA CMD NA Wild-type hfq hfq/vector N.D. N.D. 1 hfq/phfq N.D. N.D. 4 acrab acrab hfq CP, chloramphenicol; NOV, novobiocin; ACR, acriflavine; CV, crystal violet; R6G, rhodamine 6G; BENZ, benzalkonium; OXA, oxacillin; CMD, cefamandole; NA, nalidixic acid. I 3 Hfq hfq acrab μ hfq acrab hfq acrab No drug Rhodamine 6G 1 mg/l Wild-type hfq acrab Fig. 3. Hfq affects drug accumulation in E. coli. Strains wild-type, hfq and acrab were spotted onto LB agar plates containing rhodamine 6G 1 g/ml. After incubation at 37 for 20 h, E. coli colonies were observed under white light (visible waveband) 36). I 4 AcrAB Hfq hfq

4 (a) (b) Table 2. Susceptibility of E. coli strains to toxic compounds -galactosidase activity (Miller Units) Wild-type hfq acrab Promoter Relative AcrB production level (%) Fig. 4. (a) Effect of Hfq on the promoter activity of the AcrAB drug efflux system. Effect of hfq deletion on the promoter activity of the acrab operon was determined. (b) Hfq affects the production level of the AcrB multidrug efflux protein. The relative amount of AcrB production was calculated by Western blotting analysis using Science Lab 2001 Image Gauge Ver. 4.0 software 36) Wild-type hfq acrab hfq hfq acrab I 5 Hfq AcrB acrab lacz β hfq acrab hfq II 1 DsrA RNA Strain MIC (mg/l) OXA MCIPC EM R6G NOV Wild-type acrb acrb/vector acrb/pdsra acrb mdtef acrb mdtef/vector acrb mdtef/pdsra acrb rpos acrb rpos/vector acrb rpos/pdsra OXA, oxacillin; MCIPC, cloxacillin; EM, erythromycin; R6G, rhodamine 6G; NOV, novobiocin. rpos rpos σ rpos σ II 2 DsrA acrb acrb II 3 DsrA acrb μ

5 II 4 DsrA MdtEF Bright Field Fluorescence acrb/ vector acrb/ pdsra Fig. 5. Effect of dsra overexpression on drug accumulation in Escherichia coli cells. Strains acrb/vector and acrb/pdsra were spotted on LB agar plates containing 1 g/ml of ethidium bromide at a final inoculum size of 10 5 CFU/spot. After incubation, E. coli colonies were observed under white light (visible waveband, bright field) and UV light (fluorescence) 37). acrb mdtef rpos III Fold induction ratio of specific transcripts attributed to dsra overexpression (Fold increase ratio) acra acrd acre bcr cusb emra emrd emre emrk fsr maca mdfa mdta mdte mdtg mdth mdtj mdtk mdtl mdtm tolc Fig. 6. Effect of DsrA on expression levels of drug efflux and outer membrane channel genes. The level of mrna transcript was determined by qrt-pcr. The fold change ratio was calculated by dividing the expression level of the gene in the dsra-overexpressed strain by that in the parental strain 37).

6 In Escherichia coli Staphylococcus aureus Escherichia coli Vibrio harveyi Vibrio choleare Escherichia coli Escherichia coli hfq Escherichia coli Escherichia coli Escherichia coli rcsa Escherichia coli Escherichia coli

7 rpos Escherichia coli rpos Escherichia coli Escherichia coli Escherichia coli E. coli E. coli