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					 1   Induction of the MarA/soxS regulon
 2          The multiple antibiotic resistance gene (marA) and the superoxide resistance regulator
 3   (soxS) were found to be two of the most up-regulated genes in response to PMX 10070 treatment
 4   according to the microarray analysis (Fig S5). Activation of marA is known to cause low-level
 5   resistance to a wide range of antibiotics, superoxides and organic solvents (reviewed in Alekshun
 6   and Levy, 1997)(1) via the induction of efflux pumps, such as the AcrAB multidrug efflux pump.
 7   soxS is known to be induced by generation of radical oxidative species (ROS) as well as changes
 8   in the redox potential of the cytosol and induces the oxidative stress response regulon(2).
 9   Transcriptional profiling by RT-PCR confirmed a transient induction of marA (up to 14 fold in
10   polymyxin B treatment and 7.5 fold in PMX 10070 treatment; Fig S5A) and soxS (up to 75 fold
11   in polymyxin B treatment and 24 fold in PMX 10070 treatment; Supp Fig S5B). The
12   transcription of soxS is normally repressed by soxR, which binds to the polymerase binding
13   region of the soxS gene, preventing transcription. Structural changes induced by the oxidation of
14   the Fe-S cluster of soxR as a result of ROS generation or changes in the redox state of the
15   cytoplasm cause it to dissociate from the promoter, thereby initiating transcription of soxS (3).
16          Direct measurement of ROS generation in E. coli D31 was performed by using a non-
17   fluorescent, membrane permeable derivative of fluorescein, hydroxyphenyl fluorescein (HPF).
18   Once inside the cytoplasm, the phenyl group of HPF can be cleaved by reactions with hydroxyl
19   or peroxynitrite radicals (if present) to give fluorescein, which is membrane impermeable and is
20   therefore trapped in the cytoplasm. Therefore, the fluorescence emission of fluorescein can serve
21   as a reporter of ROS generation. ROS generation was measured after 3 hours of treatment with
22   MIC concentrations of PMX 10070, PMX 10072 or polymyxin B with norfloxacin (a quinilone
23   known to cause ROS generation) used as a positive control. Treatment with arylamides and
24   polymyxin showed virtually no ROS generation compared to the controls as measured by flow
25   cytometry (Supp Fig 5B). This indicates that the induction of marA and soxS is unlikely to be
26   caused by ROS generation. However, a down-regulation of the rsx regulon and rseC genes,
27   which have been shown to be involved in keeping the Fe-S cluster of soxR in a reduced state,
28   was observed in the microarray experiment. A decrease in rsx transcript abundance was
29   observed, which could explain the oxidation of soxR and subsequent induction of soxS. (Table
30   S1). Taken together, these results indicate that the activation of the oxidative-stress response
31   pathway is part of a coordinated response to the presence of antibiotic treatment.
32   Supplementary Methods:

33   ROS generation assay. A culture of E. coli D31 was grown in LB to OD600 = 0.1 and incubated
34   with 5 µM 3-(p-hydroxyphenyl) fluorescein (HPF; invitrogen) for 1 hour to allow passive
35   diffusion into cells. The cultures were then treated with appropriate concentrations of polymyxin,
36   PMX10070, PMX10072 and 250 ng/ml of norfloxacin and incubated with shaking at 37 ºC for 3
37   hours. Aliquots were then removed, pelleted and washed with PBS buffer twice and resuspended.
38   A BD FACSsort cell sorter flow-cytometer was used to measure the fluorescence intensity of
39   approximately 105 cells at 515 nm. Data analysis was performed in the BD CellQuest Pro
40   program.
41

42   Fig. S1: Arylamides permeabilized the inner-membrane slowly and less-extensively. Early log

43   phase E. coli D31 cultures were co-incubated with ONPG substrate and indicated concentrations

44   of antimicrobials. A) Polymyxin B treatment causes immediate and extensive permeabilization at

45   concentrations > 1.56 µg/mL. B) PMX 10070 treatment does not cause increased permeability of

46   ONPG substrate. C) PMX 10072 treatment causes increased ONPG permeability at high

47   treatment concentrations 40 mins after treatment.

48

49   Fig. S2: Genes down-regulated by PMX 10070 and polymyxin B treatment. Genes related to

50   protein synthesis, cell metabolism and transport are down-regulated by both treatments. More

51   genes involved in lipopolysaccharide biosynthesis and cell-cycle regulators are down-regulated

52   by PMX 10070 treatment than polymyxin B treatment.

53

54   Fig. S3: Changes in morphology caused by treatment with PMX 10070. Cultures of E. coli D31

55   were treated with 62.5 µg/mL (5X MIC) of PMX 10070 for 1 min (A), 2 min (B), 4 min (C), 8

56   min (D), 20 min (E), 40 min (F) and 60 min (G) and visualized by TEM. PMX 10070 treatment
57   shows similar changes of normal cellular morphology (increased cytoplasmic staining,

58   appearance of diffuse halo, non-uniform cellular membrane) with a slower progression than

59   treatment with PMX 10072.

60

61   Fig. S4: rcs phosphorelay is induced by arylamide treatment. CpsB-lacZ reporter strains were

62   used to measure rcs induction in response to treatment with indicated concentrations of PMX

63   10070 and PMX 10072. Polymyxin B treatment (0.39 µg/mL) was used as positive control. A)

64   Treatment with PMX 10070 induces cpsB at concentrations ≥10 µg/mL. B) Similarly, treatment

65   with PMX 10072 induces cpsB at concentrations ≥6 µg/mL. Growth attenuation caused by these

66   treatments is shown.

67

68   Fig. S5: SoxS induction is not caused by ROS generation. E. coli D31 were treated with PMX

69   10070 (8.75 µg/mL, 0.7x MIC) and polymyxin B (0.39 µg/mL, 1x MIC). RT-PCR shows the

70   time-course of the up-regulation of marA (A), and soxS (B) in response to PMX 10070 (blue)

71   and polymyxin B (red). C) There is no increase in ROS generation as a result of treatment by

72   MIC concentrations of PMX 10070, PMX 10072 or polymyxin B. Norfloxacin was used as a

73   positive control.

74

75   1.     Alekshun, M. N., and S. B. Levy. 1999. The mar regulon: multiple resistance to
76          antibiotics and other toxic chemicals. Trends Microbiol 7:410-3.
77   2.     Demple, B. 1996. Redox signaling and gene control in the Escherichia coli soxRS
78          oxidative stress regulon--a review. Gene 179:53-7.
79   3.     Koo, M. S., J. H. Lee, S. Y. Rah, W. S. Yeo, J. W. Lee, K. L. Lee, Y. S. Koh, S. O.
80          Kang, and J. H. Roe. 2003. A reducing system of the superoxide sensor SoxR in
81          Escherichia coli. EMBO J 22:2614-22.
82
83

84
85   Table S1: Down-regulation of the rsx regulon.

                                       Polymyxin B            PMX 10070

                   Gene            20 min       60 min      20 min        60 min

                    rsxA           -1.58         -1.41      -3.37         -2.03

                    rsxB           -1.13             1.06   -1.60         -1.49

                    rsxC           -1.33             1.06   -1.91         -1.96

                    rsxD           -1.22         -1.22      -1.56         -2.53

                    rsxE           -1.16         -1.44      -1.51         -1.96

                    rsxG           -1.22         -1.84      -1.94         -2.76

                    rseC           -1.08             1.05    1.18          1.62

86

				
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