1 Antibacterial Mechanism of Action of Arylamide Foldamers. 1 2 Bruk Mensa1, Yong Ho Kim1 ...

31 Pages·2011·1.32 MB·English

AAC Accepts, published online ahead of print on 15 August 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.05009-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Antibacterial Mechanism of Action of Arylamide Foldamers.
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3 Bruk Mensa1, Yong Ho Kim1, Sungwook Choi1,2,3, Richard Scott4, Gregory A. Caputo1,5 and William F.
4 DeGrado1,2,4,*.
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7 Running title: Arylamide mechanism of action o
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10 Key words: Arylamide, E. coli, whole-cell leakage, electron microscopy, DNA microarray, protein d

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11 secretion, polymyxin B, rcs ro
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14 1 Department of Biochemistry and Biophysics, University of Pennsylvania, 1010 Stellar Chance a
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15 Laboratories, 422 Curie Blvd., Philadelphia, PA 19104-4860 c
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17 2 Department of Chemistry, University of Pennsylvania, 231 S. 34th Street. Philadelphia, PA 19104- m
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20 3 current address: Chungnam National University, Daejon City, South Korea o
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22 4 PolyMedix Inc., 170 North Radnor–Chester Road, Suite 300, Radnor, PA 19087-5221
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24 5 current address: Department of Chemistry and Biochemistry, Rowan University, Glassboro NJ 08028 m
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26 * Correspondence: 2
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27 William F. DeGrado ,
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28 1009 Stellar Chance 1
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29 422 Curie Blvd. b
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30 Philadelphia, PA 19104-4860 g
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31 Tel: [phone] s
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33 [email protected]
34
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35 Abstract
36 Small arylamide foldamers designed to mimic the amphiphilic nature of antimicrobial peptides
37 have shown potent bactericidal activity against both Gram-negative and Gram-positive strains without
38 many of the drawbacks of natural AMPs. These foldamers were shown to cause large changes in the
39 permeability of the outer-membrane of Escherichia coli. They cause more limited permeabilization of
40 the inner membrane which reaches critical levels corresponding with the time required to bring about
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41 bacterial cell death. Transcriptional profiling of E. coli treated with sub-lethal concentrations of the o
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42 arylamides showed induction of genes related to membrane and oxidative stresses, with some overlap n
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43 to the effects observed for polymyxin B. Protein secretion into the periplasm and the outer-membrane a
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44 is also compromised, possibly contributing to the lethality of the arylamide compounds. The induction d

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45 of membrane-stress response regulons such as rcs coupled with morphological changes at the ro
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46 membrane observed by electron microscopy suggest that the activity of the arylamides at the
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47 membrane represents a significant contribution to their mechanism of action. p
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48 a
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49 Introduction .
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50 The development of multidrug-resistant bacteria has become an alarming health concern in m
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51 recent years. As a result, antimicrobial peptides (AMPs) have emerged as one of the leading prospects r
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52 for drug development. AMPs are retained in a wide-range of species as a first-line defense mechanism o
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53 against a broad array of microbial targets (39, 48). These peptides vary in size, sequence and efficacy, N
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54 although many share amphiphilic topologies with a charged (mostly positive) face that allows the v
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55 peptides to favorably interact with the negatively-charged bacterial membrane, and a hydrophobic face
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56 that allows for insertion into the membrane (27, 31, 32, 48, 63). This charge-interaction is thought to be r

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57 one of the fundamental properties driving the selectivity of these peptides for bacteria, which display 3
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58 negatively charged phospholipids on the outer leaflet of their membranes, as opposed to mammalian 0
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59 cells in which anionic phospholipids are primarily sequestered to the inner-leaflet (12). The abundance

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60 of phosphatidylethanolamine and lack of cholesterol in bacterial membranes has also been implicated
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61 in selectivity, presumably due to local differences in phospholipid packing and membrane curvature (2, e
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62 20, 72). Most AMPs act by causing extensive permeabilization of the membrane, characterized by
63 various proposed models such as the toroidal pore, carpet and barrel stave mechanisms, whereas others
64 have downstream cytoplasmic targets(6). There has been little documented resistance-development to
65 AMPs (47), presumably due to the membrane being the primary target (44).
66 Compounds that mimic the amphiphilic nature of AMPs such as beta-amino acid helices and
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67 antimicrobial polymers have been studied (51, 53, 62). One class of such compounds is the arylamide
68 foldamers, which consist of an arylamide backbone and various charged and hydrophobic groups
69 yielding a topographically amphiphilic structure (11, 37, 60-62). These compounds have been enhanced
70 to give significant selectivity and activity against bacteria with reduced toxicity in animal models (11).
71 Here, we investigate the mechanism of action of two arylamides; PMX 10070, which has a 2-ethyl
72 guanidinium charged substitutions, and PMX 10072, which has a 2-ethyl amine substitution on the
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73 arylamide backbone (Fig. 1A). The amphiphilic topology of these compounds is maintained by o
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74 intramolecular hydrogen bonding (11, 15). Course grained molecular dynamics simulations have n
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75 shown the equilibrium conformation of these compounds in a hydrated bilayer environment to be in the a
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76 interfacial region of the membrane, perpendicular to the bilayer normal. In these simulations, the d

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77 charged side-groups are localized to the lipid headgroup region of the bilayer while the hydrophobic ro
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78 face is inserted into the apolar lipid acyl chain region (57). These results have been corroborated
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79 experimentally using Liquid surface x-ray scattering, sum frequency generation vibrational p
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80 spectroscopy and solid-state NMR (4, 36, 60). The energetic cost for these compounds to adopt a long- a
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81 lived membrane-spanning conformation is high, rendering unlikely a mechanism which involves the .
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82 formation of stable multimeric assemblies in the membrane. Instead, transient associations involving m
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83 water, phospholipids headgroups and arylamides enhance the permeability of the membrane to solutes r
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84 and alter the surface tension of the membrane (36), possibly affecting the stability and conformation of o
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85 embedded membrane proteins. Vesicle leakage studies have shown that arylamide antimicrobials cause N
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86 less extensive permeabilization compared to naturally occurring AMPs (eg. magainin, LL-37). v
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87 However, vesicle systems are approximations of natural membranes and fail to encompass the complex
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88 asymmetric composition, modification, and regulation of bacterial membranes. Thus, it is important to r

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89 extend these studies to intact bacterial membranes (17, 32, 46). 3
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90 The induction of various genes in response to antibacterial agents, including AMP treatment, 0
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91 has been studied thoroughly in numerous Gram-negative bacteria, especially Salmonella and E. coli(38,

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92 40, 41). A number of signal transduction pathways have been implicated in AMP sensing and response,
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93 such as the rcsCB, cpxAR, baeSR, envZ/ompR and phoQP two-component systems (29, 35, 44) as well e
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94 as other transcriptional regulators including the multiple antibiotic resistance regulator (marA) (1) and
95 the superoxide stress response regulator (soxS) (14). Two-component systems have a histidine kinase
96 sensor protein in the inner membrane that, upon activation, phosphorylates a cytoplasmic response
97 regulator altering gene-expression of downstream targets (29, 58). The rcs phosphorelay and cpxAR
98 two-component systems have been shown to be strongly induced by treatments that destabilize the
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99 membrane or cause changes in osmoregulation, such as hyperosmotic shift (30), changes in
100 extracellular pH (70), Zn2+ treatment (30) and exposure to AMPs (23). The induction of these genes can
101 prove to be a powerful tool to probe the activity of these arylamides in an in vivo system. Moreover,
102 certain two-component systems have been implicated in the adaptive resistance of certain bacterial
103 species to various cationic AMPs including polymyxin B (PmB), such as the cpxA-cpxR, phoP-phoQ,
104 pmrA-pmrB and rcsB-rcsC two-component systems in E. coli (25, 67), K. pneumonae (9) and
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105 Salmonella (34, 54), and the parR-parS/phoP-phoQ two component systems in P. aeruginosa (55). o
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106 In this work, we demonstrate that the arylamide compounds affect the permeability of bacterial n
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107 membranes. Reporter gene assays coupled with direct observation of cell morphology by electron a
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108 microscopy show that arylamide treatment leads to significant disruption of the outer-membrane. This d

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109 observation is supported by recent work showing the arylamides preferentially bind to the LPS ro
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110 moieties of the cell membrane(36). Furthermore, arylamide exposure leads to increased permeability
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111 of the inner-membrane to small substrates and defects in protein translocation across the membrane. p
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112 a
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113 Materials and Methods .
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114 Antimicrobials. Arylamide foldamers were synthesized and purified as previously described (52). m
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115 Polymyxin B sulfate (Sigma) was used without further purification. r
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116 o
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117 Bacteria. N
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118 TheE. coli D31 strain used in this study was the chromosomal penicillin-V resistant isolate in the 1968 v
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119 study by Burman et al. (7). CpsB-lacZ wild type (cpsB-lacZ, SG20781) and cpsB-lacZ (cid:507)rcsF (cpzB-
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120 lacZ rcsF::kan, NM20785) strains were obtained from the Gottesman lab. CpsB-lacZ (cid:507)rcsB and cpsB- r

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121 lacZ(cid:507)rcsC strains were made by P1 transduction of rprA-lacZ (cid:507)rcsB (rprA-lacZ rcsB::kan, DH311) 3
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122 and rprA-lacZ (cid:507)rcsC strains (rprA-lacZ rcsC::kan, DH312), also obtained from the Gottesman lab, into 0
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123 the cpsB-lacZ wild type strain. E. coli DH5(cid:302) was used in protein translocation studies.
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124
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125 Time-to-kill assay. A culture of E. coli D31 was grown overnight in LB, diluted into fresh media (LB) u
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126 and grown to OD600 = 0.4. This culture was then diluted to a final concentration of 105 cells/ml into LB st
127 containing antimicrobial (12.5 μg/ml PMX 10070, 6.25 μg/ml PMX 10072 and 0.39 μg/ml polymyxin
128 B sulfate) and incubated at 37ºC with shaking. Aliquots were taken at indicated times, diluted
129 appropriately into fresh LB and plated on LB-agar plates. Colonies were counted after incubation at
130 37ºC overnight and CFU/ml in the original culture was calculated from dilution factors.
131
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132 Outer-membrane Leakage assay.E. coli D31 was grown overnight in LB supplemented with 100
133 μg/ml Ampicillin, diluted 200 fold into fresh LB supplemented with Ampicillin and grown at 37 ºC
134 with shaking to a final OD =0.2. Cells were pelleted and resuspended in equal volume PBS buffer
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135 (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate
136 monobasic, pH 7.4). To achieve desired concentrations of antimicrobial compounds, 10 μL of
137 antimicrobial stock solution (PMX compounds or polymyxin B) was added to 90 μL of resuspended
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138 cells and incubated at room temperature for indicated times. At the end of incubation period, nitrocefin o
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139 (10 mg/ml stock in DMSO; Calbiochem, EMD) was added to a final concentration of 50 μg/ml and n
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140 absorbance at 486 nm was measured every 5 seconds for 1 minute. Rate of nitrocefin hydrolysis was a
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141 calculated from the rate of hydrolysis product evolved for the first 30 seconds. As a positive control for d

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142 outer-membrane permeabilization, 10 μL of 0.1% Empigen BB detergent (EMD Chemicals) and 0.5 M ro
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143 EDTA was added to otherwise untreated cells.
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144 p
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145 Inner-membrane leakage assay. A culture of E. coli D31 was grown overnight in LB supplemented a
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146 with 1 mM IPTG, diluted 200 fold into fresh LB supplemented with IPTG and grown to a final OD600= .a
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147 0.3. In 96-well plates, 70 μL of culture was then combined with 20 μL of 4 mg/ml ONPG substrate m
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148 (Rockland) in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 10 mM MgSO4, 40 μM (cid:533)- rg
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149 mercaptoethanol) in each well. To achieve desired antimicrobial concentrations, 10 μL of 10x o
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150 antimicrobial stock solution was added for a total volume of 100 μL per well. Samples were then N
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151 incubated at 37 ºC with shaking and absorbance at 420 nm was measured every 5 minutes over a 2 hour v
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152 period. Rate of leakage was calculated from the absorbance of the ONPG hydrolysis product, o-
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153 nitrophenol in comparison to untreated samples (control). As a positive control for inner-membrane r

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154 permeabilization, 10 μL of 0.1% SDS detergent was added to otherwise untreated cells. 3
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155 0
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156 DNA microarray transcriptional profiling. A culture of E. coli D31 was grown overnight in LB and

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157 diluted 200 fold into fresh LB media and grown to a final OD600 = 0.4. The culture was then diluted 2 g
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158 fold into fresh media with antimicrobials (8.75 μg/ml PMX 10070, 0.39 μg/ml polymyxin B) in e
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159 triplicate and growth was monitored by measuring OD every 10 minutes. At 20 and 60 minutes after
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160 exposure, 1 ml culture was removed and RNA purified using Qiagen RNeasy kit according to
161 manufacturer's instructions and eluted in RNase/nucleotide free water. RNA quality was checked by
162 nano-gel assay at the Penn Microarray facility at the University of Pennsylvania. RNA was then
163 reverse-transcribed to biotin-tagged cRNA, and hybridized to Affymetrix E. coli Genome 2.0 Array
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164 gene chips and data were analyzed using Significance Analysis of Microarrays (64) at the Penn
165 microarray facility- bioinformatics group of the University of Pennsylvania, School of Medicine.
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167 RT-PCR. A culture of E. coli D31 was grown and exposed to antimicrobials identically to the
168 trancriptional profiling experiment. After exposure to antimicrobials, 5 ml culture was removed every
169 10 minutes and RNA was purified using TRIzol reagent according to manufacturer instructions. The
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170 purified RNA was dissolved in RNase/nucleotide free water and quality was checked using 260/280 o
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171 and 260/230 absorbance ratios. Samples were then digested with a DNA-free DNase treatment n
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172 (ambion) to remove DNA impurities. cDNA was synthesized using Superscript first strand synthesis a
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173 system for RT-PCR (invitrogen). cDNA levels were then quantified using Brilliant SYBR green qPCR d

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174 mastermix (stratagene) and custom primers using an MXpro 300 RTPCR instrument. MxPro-Mx3000P ro
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175 was used to determine cT values, and fold-change values for genes were computed using
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176 experimentally determined amplification efficiencies normalized to 16S RNA abundance.. p
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177 a
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178 cpsB-lacZ B-gal reporter assay. A culture of E. coli containing a chromosomal cpsB-lacZ fusion was .
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179 grown overnight in LB, diluted 200 fold into fresh LB media and grown to OD = 0.4. The culture m
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180 was diluted 2 fold into LB containing antimicrobials at indicated concentrations. Every 20 minutes, the r
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181 OD600 was measured, and 0.25 ml aliquots were removed and diluted 4 fold into Z-buffer and o
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182 permeabilized by the addition of 25 μL 0.1% SDS solution, 50 μL chloroform and vortexing. After N
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183 centrifugation, supernatant was transferred into glass test-tubes, ONPG was added to a final v
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184 concentration of 0.8 mg/ml and incubated at 37 ºC overnight. Hydrolysis was then quenched with the
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185 addition of 0.5 ml of 1 M Na2CO3, and the absorbance at 420 nm was measured to calculate miller r
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186 units (71). (cid:506)-gal reporter assays for rcs deletion strains were performed similarly. 3
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187 0
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188 TEM. A Culture of E. coli D31 was grown overnight and diluted 200 fold into fresh LB media, grown

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189 to OD600= 0.4 and diluted 2 fold into LB media with antimicrobials (final concentrations 62.5 μg/ml g
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190 PMX 10070/ PMX 10072). 1 ml aliquots were removed at indicated times, pelleted, and resuspended in e
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191 100 μL 1X HBS buffer (10 mM HEPES, 100 mM NaCl, pH 7.1). 15 μL of resuspension was dropped
192 on 400-mesh carbon grids followed by 15 μL of 1% uranyl acetate staining solution and allowed to dry.
193 Images were gathered on a FEI-Tecnai T12 Transmission Electron Microscope.
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195 TEM-1 beta-lactamase processing assay. A culture of E. coliDH5(cid:302)containing a modified pET vector
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196 with the TEM1-beta lactamase gene under its native promoter (kanr) was grown overnight in LB,
197 diluted 200 fold into fresh LB and grown to OD =0.2. Appropriate amounts of antimicrobials (25
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198 μg/ml PMX 10070, 25 μg/ml PMX 10072, 15 uM CCCP) were then added and aliquots removed at
199 indicated times. Aliquots were then pelleted, resuspended in LDS loading buffer, and loaded onto 4-
200 12% Bis-tris protein gels (invitrogen). Gels were then transferred onto nitrocellulose membranes (iBlot
201 gel transfer stacks, invitrogen) on an iBlot machine (invitrogen). The membranes were first blocked
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202 with 1% BSA in TBS-t buffer (50 mM Tris.HCl, 150 mM NaCl, 1% Tween-20) and hybridized with o
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203 primary antibody (rabbit anti-beta lactamase antibody, Chemicon) for 10 minutes, washed repeatedly n
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204 with TBS-t buffer, and then hybridized with secondary antibody (ECL anti-rabbit IgG HRP linked a
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205 whole antibody (from donkey), GE healthcare) on a SNAP ID machine (Millipore). Secondary d

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206 antibody was detected using ECL western blotting detection reagent (GE healthcare) and luminescence ro
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207 images collected on a Kodak Image Station.
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208 p
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209 The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus a
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210 (Edgaret al., 2002) and are accessible through GEO Series accession number GSE31140 .
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211 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31140). m
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212 Results
213 Antibacterial activity
214 As previously shown (11), the arylamide compounds in this study exhibit activity at low μg/mL
215 concentrations against E. coli D31 (Fig 1A). To further determine whether the arylamide antimicrobials
216 work via a bactericidal or bacteristatic mechanism, dilute cultures of E. coli (105 cells/ml) were treated
217 with MIC concentrations of antimicrobial and CFU determined at various time-points after exposure.
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218 As shown in Fig 1B, both arylamides exhibit bactericidal activity with PMX 10070 reducing viable cell o
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219 count by >99.9% at 80 min, and PMX 10072 within 60 min. In contrast, PmB caused much more rapid n
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220 bactericidal activity reducing viability by >99.9% within 10 min. a
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221 While MIC values provide a frame of reference for the activity of AMPs, the effective lethal d

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222 concentration in liquid cultures increases with the cell density of the treated culture as expected from ro
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223 an agent that needs to reach a critical concentration at the membrane. The experiments conducted
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224 below require higher cell densities than those used in MIC determination (108-109 CFU/ml, OD 0.1- tp
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225 1.0), so we investigated the effect of MIC on cell density. The effect of various concentrations of a
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226 arylamide in such conditions is illustrated in Figures 1C-1E which show the attenuation of growth of E. .
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227 coli cultures treated at early exponential phase (OD600 = 0.2) at 1x, 2x and 4x MIC concentrations of m
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228 PMX 10070, PMX 10072 and polymyxin B sulfate, a commercially available and extensively studied r
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229 cyclic antimicrobial peptide. The attenuation of growth indicates that while the compounds are o
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230 inhibitory, the cells are able to grow, albeit at a slower rate. One explanation for this effect is that at N
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231 high cell densities, larger amounts of the antimicrobials are required to reach the critical surface v
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232 concentration required for bactericidal activity.
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233 r

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234 Bacteria membrane permeabilization 3
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235 Previously, Polymyxin B has been demonstrated to cause permeabilization of the outer- and 0
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236 inner-membrane in Gram-negative bacteria (24). To determine whether the arylamide compounds also

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237 cause increased membrane permeability in bacteria, we used two independent assays to examine outer-
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238 and inner-membrane permeabilization in E. coli. Both assays rely on the differential permeability of e
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239 colorimetric enzyme substrates across the bacterial membrane in the absence or presence of membrane
240 perturbing agents. For outer-membrane permeabilization, beta-lactamase activity was measured in E.
241 coli D31. After incubation for 5, 30 and 60 minutes with arylamides, the poorly permeable beta-
242 lactamase substrate, nitrocefin, was used to probe for increased outer-membrane permeability caused
243 by treatment. The hydrolysis of the amide bond in the (cid:533)-lactam ring of the substrate by ß-lactamases
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244 (normally localized to the bacterial periplasm) results in the generation of a chromophore with
245 maximum absorbance at 486 nm. The initial kinetic rate of hydrolysis was used as a metric for outer
246 membrane permeability. Similarly, the permeability of the inner membrane was probed using the
247 cytoplasmic enzyme β-galactosidase and o-nitrophenyl-(cid:533)-D-galactopyranoside (ONPG), a colorimetric
248 substrate that can diffuse freely across the outer membrane (43) but is poorly permeable to the inner
249 membrane. The hydrolysis of ONPG liberates the chromophore o-nitrophenol, which can be monitored
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250 by its characteristic absorbance at 420 nm. Due to the slower rate of hydrolysis of ONPG as compared o
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251 to nitrocefin, we were able to incubate cultures in the presence of ONPG and were thus able to observe n
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252 both the onset and extent of leakage at various concentrations. a
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253 Upon addition of PMX 10070, PMX 10072 or PmB, a significant increase in the rate of d

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254 hydrolysis of nitrocefin (up to 30 fold over that in the absence of antimicrobials) was observed (Fig o
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255 2A), indicating a rapid and significant increase in permeability of the outer-membrane to the substrate. h
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256 The extent of permeabilization increased with longer incubation times, although this effect was less p
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257 dramatic at high concentration of arylamide treatment. This biphasic behavior has previously been a
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258 reported for other membrane active antimicrobials (17). Outer-membrane permeabilization to nitrocefin .
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259 was observed at concentrations well below the MIC for both arylamides. The inner-membrane, m
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260 however, was extensively permeabilized only by polymyxin B, with leakage observed immediately r
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261 after compound addition (Fig 2B, Supp Fig. 1A). The arylamide compounds induced very little change o
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262 in inner-membrane permeability to ONPG at most concentrations even after 2 hours of incubation (Fig N
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263 2B). Significant increases in permeability were only observed at 50 μg/ml concentration of PMX v
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264 10072 (8 fold MIC). At this concentration, increased permeability was observed to initiate at ~ 40
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265 minutes (Supp Fig 1C), which correlates with the time-to-kill observed for this compound (Fig 1B). r

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266 PMX 10070 did not show any significant increase in ONPG accessibility to the cytoplasm. We
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267 conclude that the arylamide compounds are either not able to diffuse effectively beyond the outer- 0
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268 membrane, or are largely unable to affect permeability changes to the polar ONPG substrate across the
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269 inner-membrane at the MIC.
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270 e
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271 Transmission Electron Microscopy
272 Since the arylamides seemed to cause significant outer-membrane permeabilization and a low-
273 level inner-membrane permeabilization at high concentrations of treatment, transmission electron
274 microscopy (TEM) was used to examine morphological changes that may correlate with these two
275 phenomena. A culture of E. coli D31 was treated with 62.5 μg/ml of PMX 10070 (5x MIC) and PMX
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276 10072 (10x MIC) and aliquots were removed at various times. Higher concentrations of arylamide
277 were necessary to ensure bacterial cell death at the high cell-density of the treated cultures. These
278 aliquots were resuspended , spotted on carbon grids and immediately stained with a uranyl acetate
279 solution. Uranyl acetate generally serves as a negative stain for lipid bilayers, although the UO - ion
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280 can complex with negative species such as carboxylates and phosphates and serve as a positive stain.
281 Upon treatment with PMX 10072, we observed a rapid low-level permeabilization of the inner
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282 membrane as indicated by increased accessibility of the cytoplasm to uranyl acetate, resulting in the o
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283 dark appearance of cells (Fig 3). This change was accompanied by osmotic swelling of the treated n
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284 cells. A diffuse 'halo' was also observed around many of the cells at this early timepoint. Upon a
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285 increased exposure time, these halos disappear and are followed by a transient reestablishment of d

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286 normal overall cellular shape. The membrane becomes markedly non-uniform as time progresses, with ro
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287 extensive vesiculation occurring by 20 minutes of exposure. The cells show more extensive damage to
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288 the membrane by 40 minutes and most of the cells have ruptured within 1 hour after exposure. Based p
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289 on the retention of basic cellular shape even after extensive membrane vesiculation, we hypothesize a
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290 that the change in membrane morphology happens primarily on the outer-membrane and that the .
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291 peptidoglycan and inner membrane remain relatively intact. PMX 10070 showed similar morphological m
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292 changes, albeit with a slower progression (Supp. Fig 3). A slower darkening of cells was observed, r
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293 followed by the evolution of a less intense halo that persisted for about 40 minutes. Comparable o
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294 changes in cellular morphology as a result of the disruption of the membrane were observed at the N
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295 longest exposure times for both arylamides. v
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296
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297 Beta-lactamase processing r

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298 The effect of arylamide treatment on the inner membrane is difficult to measure directly due to the 3
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299 apparent lack of permeabilization at low concentrations of treatment, as judged by substrate 0
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300 permeability assays. However, the increase in accessibility of uranyl acetate and ONPG substrate to the

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301 cytoplasm (as shown by electron microscopy and cell-leakage assays respectively) raises the question
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302 of whether the activity of these molecules at the inner-membrane contributes to their lethality. The e
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303 efficiency of protein translocation across the inner-membrane is a sensitive probe for the integrity of
304 the membrane electrochemical gradient/ proton motive force (PMF) and the proper function of the
305 membrane-associated secretion machinery. Protonophores/ionophores such as carbonyl cyanide m-
306 chlorophenylhydrazone (CCCP) have been shown to cause defects in secY-mediated secretion of
307 periplasmic and outer-membrane proteins (66).
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