Pathway-directed screen for inhibitors of the bacterial cell elongation machinery

Article abstract of DOI:10.1128/AAC.01530-18

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli. This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod– cells. A screen of 690,000 compounds identified 1,300 compounds that were active against E. coli. Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

Full text of DOI:10.1128/AAC.01530-18

AAC Accepted Manuscript Posted Online 15 October 2018
Antimicrob. Agents Chemother. doi:10.1128/AAC.01530-18
Copyright © 2018 American Society for Microbiology. All Rights Reserved.
1
2
3
A pathway-directed screen for inhibitors of the bacterial cell elongation machinery
Jackson A. Buss1, Vadim Baidin2, Michael A. Welsh1, Josué Flores-Kim1, Hongbaek Cho3, B.
McKay Wood1, Tsuyoshi Uehara1, Suzanne Walker1, Daniel Kahne2, Thomas G. Bernhardt1*
4
5
6
7
8
1Department of Microbiology and Immunobiology
Harvard Medical School
9
Boston, MA 02115
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
2Department of Chemistry and Chemical Biology
Harvard University
Cambridge, MA 02138
3Department of Biological Sciences
Sungkyunkwan University
Suwon, Gyeonggi, Korea
*To whom correspondence should be addressed:
Thomas G. Bernhardt, Ph.D.
Harvard Medical School
Department of Microbiology and Immunobiology
Boston, Massachusetts 02115
e-mail: thomas_bernhardt@hms.harvard.edu
Running title: Screen for Rod system inhibitors
Keywords: cell wall synthesis, Rod system, MreB, peptidoglycan
1

32
33
34
ABSTRACT
New antibiotics are needed to combat the growing problem of resistant bacterial infections. An
attractive avenue towards the discovery of such next generation therapies is to identify novel
inhibitors of clinically validated targets like cell wall biogenesis. We have therefore developed a
pathway-directed, whole-cell screen for small molecules that block the activity of the Rod
system of Escherichia coli. This conserved multi-protein complex is required for cell elongation
and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and
membrane proteins of unknown function that are organized by filaments of the actin-like MreB
protein. Our screen takes advantage of the conditional essentiality of the Rod system and the
ability of the beta-lactam mecillinam to cause a toxic malfunctioning of the machinery. Rod
system inhibitors can therefore be identified as molecules that promote growth in the presence
35
36
37
38
39
40
41
42
43
44
of mecillinam under conditions permissive for the growth of Rod– cells. A screen of ~690,000
45
46
47
48
49
50
51
52
identified 1,300 compounds active against E. coli. Pathway-directed screening of a majority of
this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB-
antagonist A22. Further characterization of the A22 analogs identified showed that their
antibiotic activity under conditions where the Rod system is essential was strongly correlated
with their ability to suppress mecillinam toxicity. This result combined with additional biological
studies reinforce the notion that A22-like molecules are relatively specific for MreB and
suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant
target as previously proposed.
2

53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
INTRODUCTION
New therapeutics are needed to overcome the growing problem of antibiotic resistant bacterial
infections (1). A common approach for the identification of compounds with antibacterial
activity is to screen large libraries of small molecules for potential antibiotic activity. Screens
involving in vitro assays of purified enzymes have successfully identified inhibitors of essential
bacterial targets (2). However, molecules discovered in this way often have limited activity on
cells due to their inability to penetrate the bacterial envelope and/or due to their efficient efflux
by resistance pumps (3). These issues are particularly acute for gram-negative bacteria, the
outer membrane of which poses an especially difficult barrier for drugs to cross (4). Coupled
with their efficient efflux systems that work synergistically with the outer membrane barrier,
these organisms display a high level of intrinsic antibiotic resistance.
An alternative to target-directed in vitro screens are those performed on growing bacterial cells
to identify molecules that block their replication. These screens have the advantage that
positive hits are known to have antibacterial activity from the start. However, the
disadvantages are that target identification is challenging and that many of the lethal molecules
identified are non-specific poisons with metal-chelating, detergent-like, or redox active modes-
of-action unsuitable for drug development (2, 3).
Pathway-directed, whole cell screens combine the practicality of screens performed on
growing cells with a specificity that approaches that of an in vitro assay. As with traditional
whole cell screens, this approach finds molecules with cellular activity. But, in this case,
genetic logic is used in the design such that the screening readout identifies hit molecules with
a mode-of-action that is likely limited to a particular biochemical pathway. Thus, the hits are
enriched for compounds with specific, on-target activity. Previously published screens for
3

78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
inhibitors that block wall teichoic acid (WTA) biogenesis in the gram-positive pathogen
Staphylococcus aureus and a more recent application to discover inhibitors of
lipopolysaccharide biogenesis in the gram-negative pathogen Acinetobacter baumannii are a
classic example of the pathway-directed approach (5, 6).
The classic work that demonstrated the power of pathway-directed screening relied on the
conditional essentiality of steps in wall teichoic acid (WTA) biogenesis (5). WTAs are anionic
polymers attached to the peptidoglycan (PG) cell wall of gram-positive bacteria (7). They play
important roles in the surface biology of these organisms and are essential for the virulence of
S. aureus (8). WTA polymers are built on the undecaprenol-phosphate (Und-P) lipid carrier at
the inner face of the cytoplasmic membrane. Once their synthesis is completed, they are
transported across the membrane by the flippase TarGH and then attached to the PG layer by
LCP enzymes (9, 10). Blocking the late steps of WTA synthesis is lethal due to the
accumulation of lipid-linked precursor molecules that deplete the cell of Und-P carrier that is
also needed for PG synthesis (11). However, this lethality can be suppressed by inactivating
the first enzyme in the WTA biogenesis pathway, TarO, to prevent WTA precursor
accumulation (11). Thus, a screen for molecules that kill wild-type cells but not those deleted
for the tarO gene should identify inhibitors of late stage enzymes of the WTA synthesis
pathway. Indeed, screens employing this logic successfully identified inhibitors that block the
activity of the TarGH transporter (5).
In this report, we describe the development of a pathway-directed screen for compounds that
disrupt the function of the cell elongation system of Escherichia coli. This PG biogenesis
complex is called the Rod system (a.k.a. elongasome), and it consists of five integral
membrane proteins organized by filaments of the actin-like MreB protein (12) (Fig. 1A). The
4

103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
complex is normally essential for the growth of E. coli and other rod-shaped bacteria and is
required for them to elongate their cell wall and maintain a capsule-like rod shape (13). All
components of the Rod system are conserved and broadly distributed among bacteria,
including the penicillin-binding protein called PBP2, a known beta-lactam target, and RodA,
which was recently shown to function as a cell wall polymerase (14-16). Thus, the Rod system
contains both proven and attractive new targets for therapeutic development.
As part of a broader program to identify new antibacterial agents, we screened a library of
~690,000 compounds for growth inhibitory activity against E. coli. We then counter-screened
active molecules for activity against the Rod system using our pathway-directed readouts.
Eight analogs of the MreB-antagonist A22 (17, 18) in the library were successfully identified,
validating the effectiveness of our screening approach for identifying inhibitors of at least one
component of the system. Further characterization of these inhibitors showed that their
antibiotic activity under conditions where the Rod system is essential was strongly correlated
with their ability to suppress mecillinam toxicity. This result combined with additional biological
studies reinforce the notion that A22-like molecules are relatively specific for MreB and
suggest that the lipoprotein transporter LolA is unlikely to be a physiologically relevant target of
this scaffold.
5

121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
RESULTS
Rationale for the screen
The Rod system is normally essential (19). In E. coli and other rod-shaped bacteria, depletion
of any component of the system causes cells to lose their rod-shape and develop into large
spherical cells that eventually lyse (19). However, the lethality of Rod system inactivation can
be suppressed in E. coli by moderate overproduction of the tubulin-like FtsZ protein (19) (Fig.
1B), filaments of which play a key role in the formation and activity of the cytokinetic ring
apparatus (20). We use the term FtsZUP to refer to cells producing extra FtsZ from the plasmid
pTB63. FtsZUP cells inactivated for components of the Rod system are viable and grow and
divide as small spheres rather than rods. Although the mechanism by which increased FtsZ
levels suppress Rod system essentiality is not known, the phenomenon has been useful for
genetic analysis of the machinery and studies of the effects of antibiotics on its activity (21,
22).
Several years ago, we made the surprising observation that inhibition of the Rod system
component PBP2 with a beta-lactam called mecillinam differs from genetic inactivation of the
PBP2 encoding gene (22). This led us to discover that the PBP2-mecillinam complex causes
the Rod system to malfunction and become toxic. Mecillinam-induced toxicity is caused by the
inhibition of PG crosslinking by PBP2 while glycan chains continue being produced by RodA.
Because the glycans cannot be crosslinked, they are rapidly degraded, resulting in a futile
cycle of PG synthesis and breakdown by the Rod complex (22). Blocking PG synthesis by the
Rod system in FtsZUP cells prevents the futile cycle and suppresses mecillinam toxicity (Fig.
1B). Suppression can be achieved either by genetically inactivating Rod system components
or by disrupting its activity with small molecules such as the MreB antagonist A22 (22). We
therefore reasoned that other compounds that disrupt Rod system activity by targeting MreB or
6

146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
other members of the complex (PBP2, MreC, MreD, RodA, RodZ) could be identified by
screening for molecules that suppress the toxicity of mecillinam in an FtsZUP E. coli strain (Fig.
1B).
Screening for anti-Rod system compounds
Prior to screening for Rod inhibitors, 690,000 small molecules were tested for their ability to
inhibit growth of an E. coli strain in which the lptFG genes were placed under control of the
arabinose promoter and expressed at suboptimal levels (23). These genes encode
components of the lipopolysaccharide (LPS) transport system such that the strain has a
defective outer membrane permeability barrier, which was useful to maximize the number of
lethal molecules identified. Compounds were assayed in duplicate. Endpoint absorbance
(OD600) readings for the test strain were measured following 23 hrs of growth in the presence
of compounds (Fig. 2A). Hits were identified as those molecules causing a significant reduction
(-2.5σ) in OD600 readings relative to the vehicle (DMSO) control (Fig. 2A). All commercially
available hits along with some closely related analogs were purchased and assembled into a
sub-library of 1,232 molecules to be used for identification of inhibitors for specific targets in
gram-negative pathogens.
Using this sub-library as a starting point, the screen for Rod system inhibitors was performed in
three steps: (i) screening the sub-library of lethal molecules for the ability to suppress
mecillinam toxicity in FtsZUP cells, and (ii) using additional assays to test for on-target activity
against the Rod system. We screened the sub-library for compounds capable of promoting
growth of FtsZUP E. coli cells treated with mecillinam (2.5 μg/ml, 8 x MIC) (logic based on Fig.
1B, highlighted rows). A total of 72 compounds (5.7%) were found to suppress mecillinam
toxicity (Fig. 2B), identifying them as potential Rod system inhibitors. However, prior genetic
7

171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
studies indicate that the inactivation of over 60 genes in E. coli beyond those encoding Rod
system components is capable of suppressing mecillinam toxicity (21). Thus, a range of
different targets for the hit molecules identified in the Rod screen were possible. To identify
true Rod system targeting molecules among the 72 mecillinam suppressors, we performed two
additional assays. The first assay is based on the observation that the Rod system is non-
essential in FtsZUP cells (Fig. 1B). Therefore, the lethality of a molecule that targets the Rod
system should be conditional and suppressed by elevated FtsZ levels. Of the 72 mecillinam
suppressors, 44 compounds satisfied this screen and failed to block the growth of an FtsZUP E.
coli strain with a tolC deletion to weaken its outer membrane permeability barrier (Fig. 2C).
Finally, the 44 hits from this screen were tested for their ability to alter cell shape. A total of
eight compounds were identified that induced a shape change in target cells, consistent with
the inhibition of Rod system function (Fig. 1B, 3 and 4).
Characterization of hit compounds
All eight of the hit compounds (compounds 1-8, Fig. 4B) are structurally similar to the known
MreB antagonist A22 (Fig. 4A). A22 was discovered in a screen for inhibitors of chromosome
partitioning (17) and was later found to inhibit the Rod system through a direct interaction with
MreB (18, 24, 25). Compound 1 most resembles A22 (Fig. 4B), and its anti-MreB activity was
confirmed (Table 1). The remaining seven compounds (compounds 2-8, Fig. 4B) are S-
triazine substituted analogs of the A22 scaffold reminiscent of MAC13243 (26) (Fig. 4C). This
compound was initially identified as a growth inhibitor with a lethal activity that could be
suppressed by overexpression of the lolA gene encoding an essential component of the
lipoprotein transport machinery in gram-negative bacteria (26). However, it was subsequently
found to undergo acid hydrolysis into an A22-like compound and to target MreB in
Pseudomonas aeruginosa (Fig. 4C) (27, 28, 29). Consistent with these results, we found that
8

196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
compound 4 undergoes hydrolysis, and is active, whereas the related compound 19 remains
intact and has drastically reduced A22-like cellular activity (Table 1 and Fig. S1). We therefore
infer that hit compounds 2-8 (Fig. 4B) are all likely to require hydrolysis for anti-MreB activity.
Efficacy of the screening procedure
To better understand the relative effectiveness of the screen, we wanted to know how many
total A22-like molecules were in the original library and whether any potential hits were missed
during screening. To create a comprehensive list of all molecules resembling A22 in the initial
library used for the primary screen, we performed a similarity analysis with Pipeline Pilot from
SciTegic (30) using both an extended-connectivity bit string and a feature-connectivity bit string
(FCFP). We applied a loose threshold of 0.4-0.6 to avoid false negatives (31), and identified
154 analogs (Table S1). Of these 154 compounds, 51 inhibited growth (Table S1) and 14
were acquired for the bioactive sub-library (Fig. 4B and C). An additional four A22-like analogs
were purchased for assembly of the sub-library that were not present in the initial pool. Thus,
the bioactive library used for the mecillinam suppression screen contained 18 molecules with
similarity to A22 (compounds 1-18, Fig. 4B and D).
Compounds 9-18 were not identified as having activity against the Rod system based on the
results of the sequential screening procedure (Fig. 4D). Consistent with prior SAR studies of
A22 (28, 29, 32-34), most of these molecules lack either the isothiourea or aryl halide moieties
important for bioactivity, and thus would not be predicted as screening hits. However,
compounds 9-11 resembled A22 enough to warrant further investigation of their activity.
Compound 9 effectively suppressed mecillinam toxicity, but did not induce cell rounding
(Table 1). Compounds 10 and 11, on the other hand, had weak activity that likely contributed
to their negative results in the screen. They required a concentration near or significantly
9

221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
above the screening concentration to suppress mecillinam toxicity and induce cell rounding
(Table 1). Based on the analysis of the A22-like hits from the screen and related negatives, we
conclude that the screening procedure is effective at identifying MreB antagonists and does
not suffer major issues with false negatives.
Relationship between MIC and mecillinam suppression activity of A22-like molecules
To determine the relationship between the MIC of A22-like molecules on wild-type cells and
the concentration required for mecillinam suppression, we purchased compounds 20-23 (Fig.
4E) and assayed their activities along with those of A22 and compounds 1 and 11 (Fig. 4B
and D). As expected based on prior studies of these molecules (28, 29, 32-34), they all killed
wild-type E. coli and induced a rod-to-sphere shape change (Table 1, Fig. S2). With the
exception of compound 11, which had weak activity, all of the molecules suppressed
mecillinam toxicity with a minimal concentration that was half that of their MIC. Thus, there is
good correlation between the mecillinam suppression activity and the MIC of the A22-like
molecules. Because mecillinam suppression activity is reflective of MreB antagonism, this
finding is consistent with the activity of A22-like molecules being largely (if not entirely) based
on a disruption of Rod system function at concentrations around their MIC.
LolA is unlikely to be a physiologically relevant target for A22-like molecules
The identification of MAC13243 as a potential inhibitor of LolA in the lipoprotein transport
pathway (26) and the subsequent finding that this molecule degrades into an A22-like product
has led to the suggestion that A22 and its relatives may target LolA as well as MreB (28). On
the other hand, the tight correlation between the MIC and mecillinam suppression activity
reported here supports a relatively specific mode-of-action for A22-like molecules in targeting
MreB. We therefore further investigated the chemical genetic observations connecting
10

246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
MAC13243, A22, and LolA. The finding that LolA overproduction suppressed the lethal action
of MAC13243 was the initial observation that led to the suggestion that LolA is the target for
these molecules (26). It was then later shown that LolA overproduction similarly suppressed
killing by A22, suggesting a connection between its activity and lipoprotein transport (28). An
alternative interpretation of these results comes from the prior findings that LolA
overproduction induces the Rcs envelope stress response (35), and that the Rcs stress
response provides resistance to peptidoglycan synthesis inhibitors (21, 36). Thus, we
suspected that it is the induction of the Rcs response by lolA overexpression that suppresses
the lethal activity of A22, not the overproduction of a potential target. To investigate this
possibility, we tested the effect of LolA overproduction on A22 lethality in the presence or
absence of RcsF, the lipoprotein sensor responsible for triggering the Rcs response (37). As
expected based on prior results, LolA overproduction was indeed capable of suppressing cell
killing by A22 (Fig. 5A). However, this suppressive effect was lost in ΔrcsF cells even though
similar levels of LolA were produced (Fig. 5B), indicating that suppression by LolA
overproduction requires the Rcs response.
Rod system inactivation results in an outer membrane permeability defect
MAC13243 was also recently identified in a high-throughput screen for molecules that disrupt
the outer membrane permeability barrier of E. coli (38). The authors showed that this molecule
makes cells more susceptible to bulky antibiotics like vancomycin that are normally excluded
by the outer membrane. Given the prior connection of MAC13243 with LolA (26), it was
concluded that the permeability defect induced by the compound was a result of its
interference with the lipoprotein transport pathway (38). However, based on our analysis
above, we reasoned that the observed effect of MAC13243 was more likely to result from its
breakdown product inhibiting the Rod system. Accordingly, A22 was previously shown to
11

271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
sensitize cells to novobiocin, which like vancomycin is normally excluded by the outer
membrane (39). To further investigate the effect of MreB inactivation on outer membrane
function, we compared the sensitivity of otherwise wild-type FtsZUP E. coli verses a ΔmreB
derivative growing as spherical cells. The mutant cells were found to be hypersensitive to
vancomycin and bile salts (Fig. 6). As reported previously, they were also found to be
hypersensitive to sodium dodecyl sulfate (SDS) detergent (19) (Fig. 6). We therefore conclude
that cells inactivated for the Rod system have an outer membrane permeability defect, and that
the previously observed effect of MAC13243 on cell permeability is most likely due to its
activity against MreB, not LolA.
Other potential Rod system inhibitors and their activity
In addition to A22 (17, 18) and MAC13243 (28, 29) that is hydrolyzed to form an A22-like
molecule, there are two additional antagonists of the Rod system of known structure reported
in the literature: CBR-4830 (40) and sceptrin (41). The natural product 654/A was recently
identified as a possible RodA inhibitor, but its structure has yet to be revealed (42). Of the
compounds of known structure, we wondered whether they or their close analogs were present
in the chemical library used for screening and if so, why they might not have been identified by
the assay procedure.
Sceptrin is a natural product (43) and did not have representation in our library, but similarity
analysis identified 66 potential CBR-4830 analogs in the initial library used for the lethality
screen (Table S2). Of these, 22 inhibited growth and 11 were included in our bioactive sub-
library (Table S2). None of the 11 CBR-4830 analogs suppressed mecillinam toxicity. To
further analyze why these compounds were not identified as hits, we purchased CBR-4830
and analyzed its activity in our growth assays used for screening. Consistent with previous
12

296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
results (40), we found that CBR-4830 inhibited growth of wild-type E. coli and induced cell
rounding (Table 1), suggesting that CBR-4830 may indeed target the Rod system. However,
CBR-4830 did not suppress mecillinam toxicity, nor was its inhibitory activity suppressed in
FtsZUP cells (Table 1), suggesting that CBR-4830 may have significant off-target activity.
Accordingly, CBR-4830 displayed potent lethality against an FtsZUP strain deleted for the
mreBCD operon (MIC = 12.5 μg/ml), which was only about four times greater than its MIC
against wild-type E. coli (3.1 μg/ml). Also, CBR-4830 inhibited the growth of S. aureus HG003
(MIC = 12.5 μg/mL), a gram-positive bacterium that lacks MreB (Table 1). These results are in
contrast to the relatively high specificity displayed by A22, which has a ~100-fold reduced
potency against ΔmreBCD FtsZUP cells (MIC > 100 μg/mL) relative to wild-type (MIC = 1.6
μg/mL), and is inactive against S. aureus HG003 (MIC > 100 μg/mL). Thus, we conclude that
CBR-4830 has anti-MreB activity in E. coli, but has off-target effects that are likely to prevent
the suppression of mecillinam toxicity. The potential for similar off-target activity for the CBR-
4830 derivatives in our library may also have contributed to their absence among the positive
hits from the screen.
DISCUSSION
In this report, we describe the development and implementation of a high-throughput screen
for compounds that disrupt the activity of the Rod system. The screen takes advantage of the
conditional essentiality of the complex and the ability of the beta-lactam mecillinam to make its
activity toxic (19, 22). These properties allow molecules that promote growth under special
circumstances to be identified that are also lethal against wild-type bacteria. Such a screening
process has distinct advantages over assays for lethal activity alone because it helps rapidly
eliminate non-specific toxins from the set of screening hits. Thus, downstream efforts can be
13

321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
focused on the characterization of molecules that are on-target and therefore suitable for use
as probes for studying cell wall biogenesis and/or use in potential drug development.
The Rod system as an antibiotic target
The Rod system contains several potential drug targets, the most attractive of which are the
cell wall synthase enzymes RodA and PBP2 as well as MreB. The other components (MreC,
MreD, and RodZ) are also potential targets for antibiotics, but their activities are not well
defined. Therefore, the pathway for characterizing and developing molecules active against
these proteins is likely to be more difficult than for those hitting the components with activities
that can be assayed in vitro.
One potential problem with targeting the Rod system is that its essentiality can be relatively
easily suppressed by increasing the cellular FtsZ concentration. However, the resulting
spheres grow poorly in the laboratory (19) and have outer membrane barrier defects that
would sensitize them to a range of insults inside the host. Indeed, coccoid cells of Shigella
flexneri inactivated for the Rod system also display defects in effector secretion and the ability
to invade mammalian cells (44). Thus, the activity of the Rod system is likely to be strictly
essential during infection such that the suppression observed in laboratory media is unlikely to
be problematic with respect to resistance development in the host. In addition to being
attractive targets on their own, RodA and PBP2 have paralogs (FtsW and PBP3) that perform
essential roles within the cell division apparatus. Therefore, any molecules identified that target
RodA or PBP2 have the potential to also disrupt the activity of FtsW or PBP3, respectively.
Such dual targeting activity would be ideal for antibiotic development because it would
significantly reduce potential problems with mutational resistance that can cause issues for the
development of compounds directed against a single target (45).
14

346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
An effective screening approach to identify Rod system antagonists
The successful identification of A22-like hits validates the utility of our screening procedure for
identifying antagonists of MreB. However, besides this well-studied class of molecules, no
additional inhibitor scaffolds targeting MreB or other components of the Rod system were
identified. Our previous analysis of mecillinam toxicity found that it could be readily suppressed
by the genetic inactivation of any component of the Rod system (22). Therefore, our screen
should theoretically be capable of identifying inhibitors of any member of the machinery save
for the crosslinking activity of PBP2 that is blocked by mecillinam. Why we only identified A22-
like hits targeting MreB in the screen is not known. Although we favor the idea that these
results reflect limitations in the chemical space covered by the library used for screening, an
unexpected bias of the screen for the identification of MreB antagonists cannot be ruled out
without control inhibitors active against other components of the system, of which none are
currently known.
The target of A22 and its potential as an antibiotic
A22 has been used as a probe to study MreB and Rod system function for some time (18).
The evidence for MreB being its primary target is quite strong, including mutational resistance
in the mreB gene and structures of MreB with A22 bound near its ATPase active site (18, 25,
29). However, work with the compound MAC13243 has raised questions about the specificity
of A22. MAC13243 was originally described as a potential inhibitor of LolA in the lipoprotein
transport pathway (26), but was subsequently shown to hydrolyze into an A22-like molecule
that is the active component (28). Like MAC13243, LolA overproduction was capable of
suppressing A22 lethality and LolA depletion sensitizes cells to A22, suggesting that A22 may
also target LolA.
15

371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
Although it was reasonable to link MAC13243 and A22 with LolA function based on the
chemical genetic observations (26, 28), connections based on gene overexpression and
depletion can often be indirect. In this case, LolA overproduction is known to induce the Rcs
envelope stress response (35). This observation raised the possibility that it might be the Rcs
response that is the underlying cause of the protection from compound treatment as opposed
to target overproduction. Consistent with this idea, we show here that induction of the Rcs
response is required for A22 resistance in cells overproducing LolA. Sensitization to compound
treatment upon protein depletion can be similarly indirect, especially for a factor like LolA that
plays a central role in envelope assembly. Thus, the hypersensitivity of LolA-depleted cells to
A22, which also targets envelope assembly, does not strongly support LolA targeting by A22.
A similar level of synergy was also observed between LolA depletion and the cell wall
synthesis inhibitor fosfomycin (28), which targets the precursor synthase MurA, suggesting that
LolA depletion is likely to sensitize cells to a number of treatments that disrupt cell wall
synthesis. Finally, the available biochemical data also do not provide significant support for
LolA targeting as a physiologically relevant mode-of-action for A22. The Kd for the interaction
between LolA and A22-like molecules has been measured to be 150-200 μM (28), which is far
greater than the 5.9 μM (1.6 μg/ml) MIC of A22. Finally, the ability of A22 to suppress
mecillinam toxicity and promote growth would not make sense if it also caused significant
defects in the essential lipoprotein targeting pathway. Rather, the tight correlation of MIC and
mecillinam suppression activity we observe combined with all of the available genetic and
biochemical data indicates that A22-like molecules are relatively specific for MreB.
Although A22 appears to be quite specific for MreB in bacterial cells and has been a reliable
probe for Rod system activity, it has not yet made it to the clinic as an antibacterial therapeutic.
16

396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
Problems with mammalian cell toxicity (46) as well as with high mutational resistance (2 x 10-8
for P. aeruginosa) (29) may be the issue. If these liabilities can be overcome, such A22-like
derivatives would be attractive leads for future development, either as single agents, or as a
component of a combination therapy given that Rod defects sensitize gram-negative bacteria
to other antibiotics.
Conclusion
Here, we report a screen for Rod system inhibitors. Although all of the hits identified were A22-
like molecules, only one chemical collection was screened. Despite their seemingly large size,
the chemical diversity represented in commercial collections is in actuality quite small. Genetic
selections for specific missense mutants often yield “hits” at a frequency of 10-7-10-9. Thus, it
stands to reason that even when utilizing an effective pathway-directed screen, the likelihood
of a 105- to 106-member compound library containing even one molecule that answers a
specific screen is relatively low. However, given the success of this screening procedure in
identifying MreB inhibitors, screens of additional libraries using this assay are worthwhile and
have the potential to identify new classes of anti-Rod system compounds that may be useful
for antibiotic development.
17

413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
EXPERIMENTAL PROCEDURES
Media, bacterial strains and plasmids
Cells were grown in lysogeny broth (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl ± 1.5%
BactoAgar) at 30°C unless indicated otherwise. Antibiotic concentrations used were:
Tetracycline (Tet), 5 μg/mL; Ampicillin (Amp), 50 μg/mL; Chloramphenicol (Cam), 25 μg/mL;
Kanamycin (Kan), 25 μg/mL. The bacterial strains and plasmids used in this study are listed in
Tables S3 and S4, respectively. A description of their construction is included in the
Supplementary Text along with a list of the primers used (Table S5). All deletion strains used
were obtained from the Keio knockout collection (47) and confirmed by sequencing.
We purchased A22 [S-(3,4-Dichlorobenzyl)isothiourea, HCl] from Calbiochem. We purchased
compound 1 (PH000337; 2,4-dichlorobenzyl imidothiocarbamate), compound 9 (152811;
3,4-Dichlorophenylhydrazine
imidothiocarbamate HBr,
HCl),
compound
11
(T165948;
PH012544;
4-fluorobenzyl
compound
20 (MP265,
4-chlorobenzyle
imidothiocarbamate HCl), and compound 22 (T282057; 2-chlorobenzyl imidothiocarbamate)
from Sigma. We purchased compound 4 (DP01615; 2,4-dichlorobenzyl butylamine) and
compound 21 (S01620, 3-chlorobenzyl imidothiocarbamate) from Maybridge via Fisher
Scientific. We purchased compound 10 (3-(3,4-dichlorophenyl)propan-1-amine), compound
19 (Z18380660), compound 23 ([(4-nitrophenyl)methyl]sulfanyl}methanimidamide HCl), and
CBR-4830 (EN300-214505) from Enamine. We purchased Sceptrin dihydrochloride (sc-
203258) from Santa Cruz Biotechnology. We verified the purchased compounds via LC/MS. All
compounds were dissolved in DMSO (J.T.Baker) to 10 mg/mL or the highest obtainable
concentration below 10 mg/mL, and stored at -20ºC.
18

438
439
440
441
442
443
High-throughput screening and hit validation
All screening was conducted at the ICCB-Longwood Screening Facility using 384 well plates
(Corning 3701). We performed automated pipetting with a WellMate (Agilent) or MultidropTM
Combi (Thermo Fisher), and pin transfer with a customized Epson E2C2515-UL Scara robot
paired with an Epson C3-A601S 6-axis robot arm. We screened all compounds in duplicate
plates for each condition tested.
444
445
E. coli growth inhibition screen: Each compound was pin-transferred (300 nl) into 30 μl of
LB with 1% NaCl for a final compound concentration of ≈ 25 μM. A mid-log culture of strain
NR1099 (23) (30 μl) was then added to achieve a final OD600 of 0.0002. Final concentrations of
the compounds varied based on the stock concentration of the individual libraries, but typically
fell within 6-60 μM (12-30 μg/ml). After incubation at 37ºC for ~23 hrs, the OD600 was
measured with an EnVision instrument (Perkin Elmer). Wells with an average OD600 of less
than 0.5 were designated as hits. The data were collated and a preliminary structural analysis
performed to eliminate potential PAINS compounds (48). Compounds that were annotated as
a known antibiotic (e.g. erythromycin) were also removed from the final hit list. All remaining
hits that were commercially-available were purchased as well as close analogs when possible.
Note that NR1099 only has a moderate permeability defect under the conditions used for
screening. It was found to be roughly 2-3 times as sensitive as its parent strain to novobiocin,
moenomycin, aztreonam, trimethoprim, and ciprofloxacin. Its sensitivity to rifampicin was
unchanged.
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
Mecillinam suppression screen: MG1655 [WT] with pTB63 [contains ftsQAZ operon,
pSC101 origin] was grown in LB-Tet overnight at 30ºC. The following morning, the culture was
diluted to an OD600 of 3.3 x 10-6 in LB with added 40 mM NaNO3 to serve as a terminal electron
19

463
464
465
466
467
468
469
470
471
472
473
474
475
acceptor and enhance growth in the 384-well plate format where aeration is poor. An aliquot
(30 µl) of this culture (equivalent to ~150 cells) was added to all wells of a 384-well plate
(Corning, 3701). The bioactive sub-library was then pin transferred to the plates (100 nL of
each compound). After incubation at room temperature for ~30 min, 20 μl of LB containing
6.25 μg/mL mecillinam was applied to each well, resulting in a final volume of 50 μl and a final
mecillinam concentration of 2.5 μg/mL. As above, final compound concentration varied based
on the stock concentration of the individual libraries but typically fell within 2-30 μM (4-10
μg/ml). The plates were incubated for ~72hrs at 30ºC and the OD600 was measured. The
known Rod inhibitor, A22, was used as a positive control on each plate (final OD600 = 0.42 ±
0.02, n = 128), and the compound vehicle, dimethyl sulfoxide (DMSO), was used as the
negative control (final OD600 = 0.05 ± 0.03, n = 480). Hit criteria required that both replicate
values be above a x ± 3σ cutoff (OD600 ≥ 0.158), which resulted in 72 hits (5.8%). Taking into
̅
account technical replicates, we estimate our false positive rate to be ~0.1%.
476
477
FtsZUP suppression screen: Strain JAB063 [ΔtolC] containing pTB63 was grown in LB
overnight. The resulting culture was then diluted to an OD600 of 2 x 10-6 the following morning
and 50 μl was added to each well. Hit compounds from screen # 2 were then pin transferred
(100 nl each) to the wells and the plates were incubated at 30ºC for ~48hrs before measuring
478
479
480
481
482
483
484
485
486
487
the OD600. Hits were identified as those with replicate values above a x ± 1σ cutoff (OD600
≥
̅
0.07), which resulted in 44 hits (61%). This loose cutoff allowed us to cast a wide net for
potential rod inhibitors.
Cytological screening
E. coli MG1655 ΔtolC [JAB063] was grown to mid-log phase in LB, back-diluted to an OD600 of
0.05 and applied to all wells of a 96-well plate (Corning). Potential Rod system inhibitors were
20

488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
added in a two-fold dilution series with the final concentration ranging from 0.8-25 μg/ml. Cells
were then grown with aeration at 30ºC in a VersaMax Microplate Reader with OD600
measurements taken every 5min. After treatment for 5hrs, samples at the highest sub-
inhibitory concentration were imaged as described previously (49). Morphologies were
assessed qualitatively, and those compounds inducing spherical or near spherical cell shape
identified as the final set of hits. In addition to the 44 hits identified as putative Rod inhibitors in
screen #3, the remaining 28 compounds were also assessed cytologically. All 28 compounds
failed to induce cell rounding.
Compound validation
Compounds 1, 4, 9-11, 19-24, CBR-4830 and Sceptrin were purchased (2-100 mg) and
validated for growth inhibition via broth microdilution in 96- and 384-well plates (Corning) using
the same growth conditions as described for Screen #2. We also validated mecillinam
suppression and suppression by FtsZUP over a range of concentrations (0.2-100 μg/ml) using
an HP D300 dispenser for precise titration and a MultidropTM Combi (Thermo Fisher) for
automated pipetting. Bright-field microscopy was performed on wild-type MG1655 cells after
treatment with 10-100 μg/ml of compound for 3-4 hrs at 30ºC.
Viability assays
Wild-type E. coli MG1655 and ΔrcsF [HC397] containing either an empty vector (pHC800) or
lolA expression construct (pJAB107) were grown to an OD600 of 1.0, serial diluted ten-fold and
5 μl of each dilution spotted onto LB agar containing 0, 1.25, 2.5, 5 or 10 μg/ml A22 in the
presence or absence of 500 μM IPTG. Plates were incubated at 30oC for 48hrs and
photographed. Similarly, wild-type E. coli MG1655 and ΔmreB [TU233] expressing FtsZUP
(pTB63) were grown to mid-log phase, diluted to an OD600 of 0.1, serial diluted ten-fold and
21

513
514
515
516
517
518
519
520
521
522
523
524
525
526
spotted onto MacConkey agar or LB agar supplemented with 10 μg/ml of vancomycin, 50
μg/ml of vancomycin, or 1% sodium dodecyl sulfate (SDS). Plates were incubated at 30oC for
48hrs and photographed.
Compound hydrolysis and LC-MS analysis
Stocks of compounds 4 and 19 were diluted to 500 μM in water with 5% acetonitrile and 0.1%
trifluoroacetic acid (TFA). This solution was allowed to sit at room temperature for 0.5 or 18 hr
before LC-MS analysis. LC-MS data was collected using an Agilent 6120 Quadrupole LC-MS
using electrospray ionization (ESI). LC-MS was conducted with the MS operating in positive
ion mode. The compound solutions (5 μL injections) were separated on a Waters Symmetry
Shield RP18 column (5 μm, 3.9 x 150 mm) using the following method: flow rate 0.5 mL/min,
95% solvent A (H2O, 0.1% formic acid) for 5 min followed by a linear gradient of 5% solvent B
(acetonitrile, 0.1% formic acid) to 100% B over 15 min.
22

527
528
529
530
531
532
533
534
ACKNOWLEDGEMENTS
The authors would like to thank all members of the Bernhardt, Rudner, Kahne and Walker labs
for advice and helpful discussions. We also thank the staff at The Institute for Chemistry and
Cell Biology (ICCB-Longwood) Screening Facility at Harvard Medical School for their help in
screening and data analysis, and the Microscopy Resources on North Quad (MicRoN) imaging
core at Harvard Medical School. This work was supported by the National Institutes of Health
(CETR U19 AI109764 to TGB, DK, and SW; F32GM123579 to MAW; F32AI36431 to JFK).
23

535
536
537
538
539
540
541
REFERENCES
1.
Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini
C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J,
Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N,
Theuretzbacher U, Magrini N, WHO Pathogens Priority List Working Group. 2018.
Discovery, research, and development of new antibiotics: the WHO priority list of
antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18:318–327.
542
543
2.
3.
4.
5.
Silver LL. 2011. Challenges of antibacterial discovery. Clinical Microbiology Reviews
24:71–109.
544
545
Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL. 2007. Drugs for bad bugs:
confronting the challenges of antibacterial discovery. Nat Rev Drug Discov 6:29–40.
546
547
Silver LL. 2016. A Gestalt approach to Gram-negative entry. Bioorganic & Medicinal
Chemistry 24:6379–6389.
548
549
550
551
Swoboda JG, Meredith TC, Campbell J, Brown S, Suzuki T, Bollenbach T,
Malhowski AJ, Kishony R, Gilmore MS, Walker S. 2009. Discovery of a small
molecule that blocks wall teichoic acid biosynthesis in Staphylococcus aureus. ACS
Chem Biol 4:875–883.
552
553
554
555
6.
Zhang G, Baidin V, Pahil KS, Moison E, Tomasek D, Ramadoss NS, Chatterjee AK,
McNamara CW, Young TS, Schultz PG, Meredith TC, Kahne D. 2018. Cell-based
screen for discovering lipopolysaccharide biogenesis inhibitors. Proc Natl Acad Sci USA
115:6834–6839.
24

556
557
7.
8.
Brown S, Santa Maria JP, Walker S. 2013. Wall teichoic acids of gram-positive
bacteria. Annu Rev Microbiol 67:313–336.
558
559
560
561
Wanner S, Schade J, Keinhörster D, Weller N, George SE, Kull L, Bauer J, Grau T,
Winstel V, Stoy H, Kretschmer D, Kolata J, Wolz C, Bröker BM, Weidenmaier C.
2017. Wall teichoic acids mediate increased virulence in Staphylococcus aureus. Nat
Microbiol 2:16257.
562
563
9.
Schaefer K, Matano LM, Qiao Y, Kahne D, Walker S. 2017. In vitro reconstitution
demonstrates the cell wall ligase activity of LCP proteins. Nat Chem Biol 13:396–401.
564
565
566
567
10.
Kawai Y, Marles-Wright J, Cleverley RM, Emmins R, Ishikawa S, Kuwano M, Heinz
N, Bui NK, Hoyland CN, Ogasawara N, Lewis RJ, Vollmer W, Daniel RA, Errington
J. 2011. A widespread family of bacterial cell wall assembly proteins. The EMBO
Journal 30:4931–4941.
568
569
570
571
11.
D'Elia MA, Pereira MP, Chung YS, Zhao W, Chau A, Kenney TJ, Sulavik MC, Black
TA, Brown ED. 2006. Lesions in teichoic acid biosynthesis in Staphylococcus aureus
lead to a lethal gain of function in the otherwise dispensable pathway. Journal of
Bacteriology 188:4183–4189.
572
573
12.
13.
Shi H, Bratton BP, Gitai Z, Huang KC. 2018. How to Build a Bacterial Cell: MreB as
the Foreman of E. coli Construction. Cell 172:1294–1305.
574
575
576
Typas A, Banzhaf M, Gross CA, Vollmer W. 2011. From the regulation of
peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Micro 10:123–
136.
25

577
578
579
14.
15.
Meeske AJ, Riley EP, Robins WP, Uehara T, Mekalanos JJ, Kahne D, Walker S,
Kruse AC, Bernhardt T, Rudner DZ. 2016. SEDS proteins are a widespread family of
bacterial cell wall polymerases. Nature 537:634–638.
580
581
582
Cho H, Wivagg CN, Kapoor M, Barry Z, Rohs PDA, Suh H, Marto JA, Garner EC,
Bernhardt T. 2016. Bacterial cell wall biogenesis is mediated by SEDS and PBP
polymerase families functioning semi-autonomously. Nat Microbiol 1:16172.
583
584
16.
17.
Zhao H, Patel V, Helmann JD, Dörr T. 2017. Don't let sleeping dogmas lie: new views
of peptidoglycan synthesis and its regulation. Mol Microbiol 106:847–860.
585
586
587
588
Iwai N, Nagai K, Wachi M. 2002. Novel S-benzylisothiourea compound that induces
spherical cells in Escherichia coli probably by acting on a rod-shape-determining
protein(s) other than penicillin-binding protein 2. Biosci Biotechnol Biochem 66:2658–
2662.
589
590
18.
19.
Gitai Z, Dye NA, Reisenauer A, Wachi M, Shapiro L. 2005. MreB actin-mediated
segregation of a specific region of a bacterial chromosome. Cell 120:329–341.
591
592
593
Bendezú FO, de Boer PA. 2008. Conditional Lethality, Division Defects, Membrane
Involution, and Endocytosis in mre and mrd Shape Mutants of Escherichia coli. Journal
of Bacteriology 190:1792–1811.
594
595
20.
21.
Wagstaff J, Löwe J. 2018. Prokaryotic cytoskeletons: protein filaments organizing
small cells. Nature Publishing Group 16:187–201.
596
597
598
Lai GC, Cho H, Bernhardt T. 2017. The mecillinam resistome reveals a role for
peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.
PLoS Genet 13:e1006934.
26

599
600
22.
23.
Cho H, Uehara T, Bernhardt T. 2014. Beta-lactam antibiotics induce a lethal
malfunctioning of the bacterial cell wall synthesis machinery. Cell 159:1300–1311.
601
602
603
Yao Z, Davis RM, Kishony R, Kahne D, Ruiz N. 2012. Regulation of cell size in
response to nutrient availability by fatty acid biosynthesis in Escherichia coli. Proc Natl
Acad Sci USA 109:E2561–8.
604
605
606
24.
Bean GJ, Flickinger ST, Westler WM, McCully ME, Sept D, Weibel DB, Amann KJ.
2009. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a
low-affinity state in MreB. Biochemistry 48:4852–4857.
607
608
25.
26.
van den Ent F, Izoré T, Bharat TA, Johnson CM, Löwe J. 2014. Bacterial actin MreB
forms antiparallel double filaments. Elife 3:e02634.
609
610
611
Pathania R, Zlitni S, Barker C, Das R, Gerritsma DA, Lebert J, Awuah E, Melacini
G, Capretta FA, Brown ED. 2009. Chemical genomics in Escherichia coli identifies an
inhibitor of bacterial lipoprotein targeting. Nat Chem Biol 5:849–856.
612
613
614
27.
28.
Takacs CN, Poggio S, Charbon G, Pucheault M, Vollmer W, Jacobs-Wagner C.
2010. MreB drives de novo rod morphogenesis in Caulobacter crescentus via
remodeling of the cell wall. Journal of Bacteriology 192:1671–1684.
615
616
617
618
Barker CA, Allison SE, Zlitni S, Nguyen ND, Das R, Melacini G, Capretta AA,
Brown ED. 2013. Degradation of MAC13243 and studies of the interaction of resulting
thiourea compounds with the lipoprotein targeting chaperone LolA. Bioorg Med Chem
Lett 23:2426–2431.
27

619
620
621
29.
Yamachika S, Sugihara C, Tsuji H, Muramatsu Y, Kamai Y, Yamashita M. 2012.
Anti-Pseudomonas aeruginosa compound, 1,2,3,4-tetrahydro-1,3,5-triazine derivative,
exerts its action by primarily targeting MreB. Biol Pharm Bull 35:1740–1744.
622
623
30.
31.
32.
Xia X, Maliski EG, Gallant P, Rogers D. 2004. Classification of kinase inhibitors using
a Bayesian model. J Med Chem 47:4463–4470.
624
625
Rogers D, Hahn M. 2010. Extended-connectivity fingerprints. J Chem Inf Model
50:742–754.
626
627
628
Iwai N, Ebata T, Nagura H, Kitazume T, Nagai K, Wachi M. 2004. Structure-activity
relationship of S-benzylisothiourea derivatives to induce spherical cells in Escherichia
coli. Biosci Biotechnol Biochem 68:2265–2269.
629
630
631
33.
34.
Iwai N, Fujii T, Nagura H, Wachi M, Kitazume T. 2007. Structure-activity relationship
study of the bacterial actin-like protein MreB inhibitors: effects of substitution of benzyl
group in S-benzylisothiourea. Biosci Biotechnol Biochem 71:246–248.
632
633
634
635
636
Nicholson A, Perry JD, James AL, Stanforth SP, Carnell S, Wilkinson K, Anjam
Khan CM, De Soyza A, Gould FK. 2012. In vitro activity of S-(3,4-
dichlorobenzyl)isothiourea hydrochloride and novel structurally related compounds
against multidrug-resistant bacteria, including Pseudomonas aeruginosa and
Burkholderia cepacia complex. Int J Antimicrob Agents 39:27–32.
637
638
639
35.
Chen MH, Takeda S, Yamada H, Ishii Y, Yamashino T, Mizuno T. 2001.
Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved
in capsular synthesis in Escherichia coli. Biosci Biotechnol Biochem 65:2364–2367.
28

640
641
642
36.
37.
Laubacher ME, Ades SE. 2008. The Rcs phosphorelay is a cell envelope stress
response activated by peptidoglycan stress and contributes to intrinsic antibiotic
resistance. Journal of Bacteriology 190:2065–2074.
643
644
645
646
Cho S-H, Szewczyk J, Pesavento C, Zietek M, Banzhaf M, Roszczenko P, Asmar A,
Laloux G, Hov A-K, Leverrier P, Van der Henst C, Vertommen D, Typas A, Collet J-
F. 2014. Detecting envelope stress by monitoring β-barrel assembly. Cell 159:1652–
1664.
647
648
649
38.
Muheim C, Götzke H, Eriksson AU, Lindberg S, Lauritsen I, Nørholm MHH, Daley
D. 2017. Increasing the permeability of Escherichia coli using MAC13243. Sci Rep
7:17629.
650
651
39.
40.
Taylor PL, Rossi L, De Pascale G, Wright GD. 2012. A forward chemical screen
identifies antibiotic adjuvants in Escherichia coli. ACS Chem Biol 7:1547–1555.
652
653
654
Robertson GT, Doyle TB, Du Q, Duncan L, Mdluli KE, Lynch AS. 2007. A Novel
indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a
substrate for MexAB-OprM. Journal of Bacteriology 189:6870–6881.
655
656
41.
42.
Rodríguez AD, Lear MJ, La Clair JJ. 2008. Identification of the binding of sceptrin to
MreB via a bidirectional affinity protocol. J Am Chem Soc 130:7256–7258.
657
658
659
Emami K, Guyet A, Kawai Y, Devi J, Wu LJ, Allenby N, Daniel RA, Errington J.
2017. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic
discovery for the peptidoglycan polymerase pathway. Nat Microbiol 2:16253.
660
661
43.
Walker RP, Faulkner DJ, Van Engen D, Clardy J. 1981. Sceptrin, an antimicrobial
agent from the sponge Agelas sceptrum. J Am Chem Soc 103:6772–6773.
29

662
663
664
44.
Noguchi N, Yanagimoto K, Nakaminami H, Wakabayashi M, Iwai N, Wachi M,
Sasatsu M. 2008. Anti-infectious effect of S-benzylisothiourea compound A22, which
inhibits the actin-like protein, MreB, in Shigella flexneri. Biol Pharm Bull 31:1327–1332.
665
666
45.
46.
Silver LL. 2007. Multi-targeting by monotherapeutic antibacterials. Nat Rev Drug
Discov 6:41–55.
667
668
669
van Teeseling MCF, de Pedro MA, Cava F. 2017. Determinants of Bacterial
Morphology: From Fundamentals to Possibilities for Antimicrobial Targeting. Front
Microbio 8:1264.
670
671
672
47.
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Datsenko KA, Tomita M, Wanner
BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout
mutants: the Keio collection. Mol Syst Biol 2.
673
674
48.
49.
50.
Baell J, Walters MA. 2014. Chemistry: Chemical con artists foil drug discovery. Nature
513:481–483.
675
676
Buss JA, Peters NT, Xiao J, Bernhardt T. 2017. ZapA and ZapB form an FtsZ-
independent structure at midcell. Mol Microbiol 104:652–663.
677
678
679
Spratt BG. 1975. Distinct penicillin binding proteins involved in the division, elongation,
and shape of Escherichia coli K12. Proceedings of the National Academy of Sciences
72:2999–3003.
680
681
682
51.
Bendezú FO, Hale CA, Bernhardt T, de Boer PA. 2009. RodZ (YfgA) is required for
proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. The EMBO
Journal 28:193–204.
30