bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 L-arabinose induces the formation of viable non-proliferating Vibrio
2 cholerae spheroplasts
3
4 Elena Espinosa1#, Sandra Daniel1#, Sara B. Hernández2, Felipe Cava2, François-Xavier
5 Barre1@, Elisa Galli1@
6
7 1 Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS,
8 Gif-sur-Yvette, France
9 2 The laboratory for Molecular Infection Medicine Sweden (MIMS), Department of
10 Molecular Biology, Umeå University, Umeå, Sweden
11
12 # Contributed equally to the work
13 @ Address correspondence to: [email protected],
15
16
17
18
19
20
21
22
23
24
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
26 Abstract
27 A general survival strategy of many life forms faced with harmful growth conditions is to enter
28 into a non-proliferating state until conditions suitable for growth are restored. In bacteria, this
29 survival strategy is associated with antimicrobial tolerance, chronic infections and
30 environmental dispersion. In particular, the agent of the deadly human disease cholera, Vibrio
31 cholerae, undergoes a morphological transition from a rod-shaped proliferative form to a
32 spherical non-proliferating form after exposure to cold or cell wall targeting antibiotics. Growth
33 is resumed when the adverse conditions have ceased.
34 Here, we show that a component of the hemicellulose and pectin of terrestrial plants, L-
35 arabinose, triggers the formation of non-proliferating V. cholerae spherical cells, which are able
36 to return to growth when L-arabinose is removed from the growth medium. We found that the
37 cell wall of L-arabinose treated V. cholerae cells has a peptidoglycan composition similar to
38 the cell wall of spheroplasts and that they revert to a wild-type morphology through the
39 formation of branched cells like L-forms. Unlike L-forms, however, they are osmo-resistant.
40 Through a random Tn genetic screen for mutants insensitive to L-arabinose, we identified genes
41 involved in the uptake and catabolism of galactose and in glycolysis. We hypothesize that L-
42 arabinose is enzymatically processed by the galactose catabolism and glycolysis pathways until
43 it is transformed in a product that cannot be further recognized by V. cholerae enzymes.
44 Accumulation of this enzymatic by-product triggers the formation of viable non-dividing cell
45 wall deficient spherical cells.
46
47
48 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
49 Introduction
50 Cholera is an acute diarrhoeal disease caused by ingestion of food or water contaminated with
51 Vibrio cholerae, a curved rod shape bacterium that propagates in warm briny and salty waters.
52 Cold stops V. cholerae proliferation. However, the bacterium has the ability to persist for
53 months in cold water under the form of a spheroplast, i.e. a cell wall deficient spherical cell,
54 and return to growth when the sea temperature rises [1–3]. Similarly, V. cholerae is known to
55 persist under a spherical form in biofilms [4]. Correspondingly, it was observed that V. cholerae
56 tolerates exposure to antibiotics inhibiting cell wall synthesis [5].
57 The high propagation rate of V. cholerae and its capacity to survive unfavourable growth
58 conditions have led to several pandemics, which have caused and are still causing major socio-
59 economic perturbations [6]. Seven cholera pandemics have been recorded since the beginning
60 of the 18th century. The 7th pandemic started over 50 years ago and is still ongoing. V. cholerae
61 strains can be grouped into 12 distinct lineages, out of which only one gave rise to pandemic
62 clones [7]. Transition from the previous (6th) to the current (7th) pandemic was associated with
63 a shift between 2 different phyletic subclades, the so-called classical and El Tor biotypes [7–
64 11]. Isolates of the current pandemic are rapidly drifting and it is suspected that the constant
65 appearance of new atypical pathogenic variants of V. cholerae will eventually lead to a more
66 virulent strain that will start a new (8th) pandemic. This motivated extensive research on the
67 physiology of the bacterium and its evolution towards pathogenicity [7–11].
68 Inducible promoters permit to study the phenotypes of null mutations of essential genes and/or
69 to trigger the production of potentially toxic recombinant protein fusions at specific stages of
70 the cell cycle. In particular, a tightly-regulated high-level expression inducible system based on
71 the promoter of the Escherichia coli araBAD operon and its regulator araC, known as the PBAD
72 system [12], was and still is instrumental to many V. cholerae studies. The AraBAD enzymes
73 allow E. coli to exploit L-arabinose (L-Ara), a component of the hemicellulose and pectin of bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
74 terrestrial plants, as a carbon and energy source [13]. AraC acts both as a positive and a negative
75 regulator, repressing PBAD in the absence of L-Ara and activating its transcription when bound
76 to it [13]. V. cholerae lacks a bona fide arabinose import and metabolization pathway.
77 Nevertheless, the E. coli PBAD system proved to be very effective in V. cholerae, which
78 suggested that L-Ara was imported in the cytoplasm of the cells. However, we and others
79 recently reported that L-Ara could interfere with the growth of the bacterium, calling for a better
80 understanding of the impact of L-Ara on the physiology of V. cholerae and the pathway leading
81 to its toxicity [14].
82 Here, we show that V. cholerae cells stop dividing or elongating and lose their characteristic
83 curved rod cell shape in the presence of >0.5% (w/v) and >0.1% (w/v) of L-Ara in rich and
84 poor media, respectively. V. cholerae cells become spherical and morphologically akin to the
85 spheroplasts obtained by exposure to cold temperatures [1,2,15] or cell wall targeting
86 antibiotics [5]. We found that the spheroplasts are able to revert to exponentially growing rods
87 in only a few generations when L-Ara is removed, demonstrating that they remain viable. We
88 further show that L-Ara is imported by the galactose transport system and that it interferes with
89 the metabolism of V. cholerae by entering the galactose and glycolysis catabolic pathways.
90 Finally, we show that V. cholerae mutants with impaired physiology are more sensitive to the
91 presence of L-Ara, morphologically transitioning to spheroplasts with as little as 0.01% (w/v)
92 of L-Ara in poor media. Taken together, these results suggest that spheroplasts formation might
93 be a general survival strategy of Vibrios when faced with conditions that perturb their
94 metabolism. From a technical point of view, they indicate how the PBAD expression system can
95 still be used in V. cholerae without perturbing the metabolism of the bacterium.
96
97
98 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
99 Results
100 L-arabinose induces the formation of non-dividing spherical cells
101 To study the effect of L-Ara on cell morphology and growth, we added increasing
102 concentrations of L-Ara to wild-type N16961 V. cholerae cells under exponential growth in
103 different liquid media. Cells grown in M9-MM appeared with a wild-type rod shape in the
104 absence or up to 0.02% (w/v) L-Ara, but the entire cell population became spherical in the
105 presence of 0.1% (w/v) L-Ara (Figure 1A). In cultures grown in M9-MM supplemented with
106 casamino acids (CAA), spherical cells started to appear at 0.2% (w/v) of L-Ara. At 0.5% (w/v)
107 of L-Ara, all cells exhibited a spherical morphology. In LB, the concentration of L-Ara had to
108 be increased to 1% (w/v) to induce loss of the normal cell shape (Figure1A). The morphological
109 appearance of L-Ara treated cells resembles that of non-proliferative cells obtained by
110 incubating V. cholerae cells at 4C [2,4] or by treating them with cell wall targeting antibiotics
111 [5] (Figure 1B).
112 In parallel to microscopic inspection, we followed the optical density of cell cultures over time.
113 In all tested media, L-Ara had a detrimental effect on cell proliferation at the same
114 concentrations at which loss of cell morphology was observed (Supplementary Figure 1).
115 Additional sugars or carbon sources were tested for similar effects on cell shape and growth,
116 but none of them, including D-arabinose, affected V. cholerae growth or morphology (Table 1
117 and Supplementary Figure 2).
118 Mutants with impaired physiology are more sensitive to L-Ara
119 Strikingly, we observed that physiologically impaired V. cholerae mutants were more sensitive
120 to the presence of L-Ara. In particular, the presence of as little as 0.01% (w/v) of L-Ara was
121 sufficient to induce the formation of non-dividing spherical cells in a N16961 strain carrying
122 two copies of the ssb gene, which codes for an essential single strand DNA binding protein
123 implicated in the regulation of replication, transcription and homologous recombination repair bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
124 [16] (Figure 1C). Likewise, the presence of 0.01% (w/v) of L-Ara was sufficient to induce the
125 formation of non-dividing spherical cells in MCH1, a mono-chromosomal V. cholerae strain,
126 when the SlmA nucleoid occlusion protein was overproduced [17] (Figure 1C).
127 Transition dynamics to spherical cells at the population level
128 To visually inspect the morphological transition at the population level over time, we collected
129 cell samples every hour for 10 hours after L-Ara addition and examined them at the microscope.
130 Cells were divided in three categories based on their shape: cells with a rod shape, cells
131 composed of a rod and a small or large irregular bulge protruding from the cell wall, which we
132 refer to as bleb, and cells with a spherical shape (Figure 2A). Spherical cells started to appear
133 after 5 hours and comprised 90% of the cell population after 9 hours. The sharp increase in the
134 proportion of spherical cells in the population corresponded to an equally fast decline in rod
135 shaped cells, which dropped to less than 10% of the cell population at the end of the experiment.
136 Cells composed of a rod and a bleb appeared around 4 hours after L-Ara addition. Blebs were
137 randomly located on the surface of the cells. In particular, there was no preference for mid-cell
138 or cell pole locations (Supplementary Figure 3). Cells with protruding blebs never represented
139 more than 5% of the entire cell population and almost disappeared at the end of the time course,
140 which suggested that they corresponded to a transient state between the rod and the spherical
141 state (transitioning cells). Taken together, these results suggest that a few hours are required
142 after the growth arrest before morphological transition. However, once transition is initiated the
143 formation of spherical cells is very fast.
144 The size of the spherical cells was heterogeneous (Figure 1). The diameter of the majority of
145 cells was comprised between 1.5 and 1.8 μm (Figure 2B). The average of the diameter did not
146 change over time, suggesting that spherical cells neither decreased nor increased in volume
147 after their formation (Figure 2C). Based on the average cell diameter of spherical cells and the bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
148 average cell length and width of rod-shaped cells, we estimate that the cell volume of spherical
149 cells is around 2.5 times bigger than that of exponentially growing rod cells.
150 Transition dynamics to spherical cells at the single cell level
151 We performed time-lapse video-microscopy experiments to inspect the transition process from
152 rod to sphere at the single cell level (Figure 2D and Movie 1). All the observed cells displayed
153 the same transition pattern. After exposure to L-Ara, a single bleb appeared at the bacterial cell
154 surface. As the bleb increased in size, the original cell was assimilated into the forming sphere
155 until the original rod shape was completely lost. The time when a bleb became visible on the
156 cell surface varied from cell to cell. However, once started, completion of the morphological
157 change was comparable in all the cells. On an agarose M9-MM pad, blebbing cells transitioned
158 to spheres in around 2 to 3 hours. We could observe rare events in which cells failing to
159 transition to spheres lysed (Movie 2).
160 L-Ara induced spherical cells are cell wall deficient
161 The spherical shape of L-Ara treated cells reminded that described for cell wall deficient
162 (CWD) forms that have completely (protoplast-type) or almost entirely (spheroplast-type) lost
163 the peptidoglycan (PG) layer, which suggested a process of cell wall degradation or PG
164 remodelling mechanism [18]. To verify this point, we compared the PG content and
165 composition of exponentially growing V. cholerae cells and L-Ara induced non-dividing
166 spherical cells (Table 2). To limit contamination by the PG of cells that had not completely
167 transformed into spheres in the presence of L-Ara, we used MCH1 cells expressing an
168 additional copy of SlmA because of their increased sensitivity to L-Ara (Figure 1C).
169 The PG of exponentially growing and L-Ara treated cells that had completely transitioned to
170 spheres was extracted and submitted to UPLC analysis (Supplementary Figure 4A). The area
171 of the UPLC profiles showed that the amount of PG per cell in L-Ara treated bacteria was
172 around 10 times lower than the amount present in rod-shaped cells (Supplementary Figure 4B). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
173 In addition, we found that glycan chains were twice shorter in L-Ara treated cells than in
174 untreated cells. The length of glycan chains is calculated based on the number of anhydro-
175 muropeptides [19], which result from the activity of lytic transglycosylases [20,21]. Therefore,
176 a reduction in the average glycan chain length suggests an increase of lytic transglycosylase
177 activity in the presence of L-Ara. Finally, we observed that the amount of DAP-DAP cross-
178 linked muropeptides significantly increased in L-Ara treated cells. Taken together, these results
179 indicate that L-Ara induced spherical cells are spheroplasts.
180 L-Ara induced spheroplasts are viable and can revert to proliferation
181 To evaluate if L-Ara had a detrimental effect on cell viability we estimated the number of viable
182 bacteria in a time course experiment. An initial culture was split in two after 2 hours of growth
183 and L-Ara added to one of the two halves. The number of viable cells in the cultures was
184 determined by plating aliquots on LB plates and counting the numbers of colonies. Viable cell
185 count kinetics (represented as colony forming units, CFU) showed that L-Ara addition had an
186 immediate inhibitory effect on cell proliferation but did not cause a corresponding decline in
187 cell viability (Figure 3A). Indeed, 75% of the number of cells before L-Ara addition gave rise
188 to colonies after an incubation of 10 hours with L-Ara (Figure 3B).
189 Time-lapse video-microscopy was performed to determine how non-proliferating spherical
190 cells could return to proliferation and recover a curved rod shape after L-Ara removal. Single
191 cell analyses showed that reversion to proliferating rods started with the elongation of the
192 spheroplasts, which was followed by the formation of multiple protrusions on their surface
193 (Figure 3C and Movie 3). The protrusions elongated outward, giving rise to multiple branched
194 cells, and the original rod shape was recovered after only a few division events. The time
195 required to initiate elongation greatly differed from cell to cell: the recovery process of some
196 cells started almost immediately after the removal of L-Ara and took a few hours for other cells.
197 However, the length of time between the initiation of the recovery process and its completion bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
198 was similar for all the cells. On an agarose M9-MM pad, cells transitioned from elongating
199 spheres to symmetrically dividing rods in around 4 to 5 hours.
200 Time-lapse video-microscopy observations further suggested that the overall 25% loss of CFU
201 after 10 hours of L-Ara treatment (Figure 3B) was accounted for by the number of cells that
202 lysed during the transition to spheroplasts after the addition of L-Ara (Movie 2) and by those
203 that lysed during the recovery process after L-Ara removal (Movie 4). As no osmo-protectant
204 was added to the growth media, this observation suggests that the spheroplasts are osmo-
205 resistant.
206 wigKR is essential for the recovery of cells after L-Ara treatment
207 The histidine kinase/response regulator pair wigKR is thought to induce a higher expression of
208 the full set of cell wall synthetic genes in response to cell wall damages [22]. It was previously
209 reported that wigKR was essential for the recovery of the cell shape and the return to
210 proliferation of V. cholerae cells treated with cell wall targeting antibiotics [22]. In the presence
211 of L-Ara, wigKR cells formed spheroplasts at the same timing than wild-type V. cholerae cells
212 and blebs remained distributed all around the surface of cells (Supplementary Figure 5A).
213 However, reversion was dramatically different. After removal of L-Ara, wigKR spheroplasts
214 immediately started to grow in diameter, expanding continuously in size until they exploded
215 (Figure 3D and Movie 5). On the contrary, wigKR cells that had stopped dividing but had not
216 yet transitioned to spheroplasts were able to return to a proliferative state without any obvious
217 defect (Movie 6). After 10 hours incubation with L-Ara, only 10% of the wigKR cells could
218 still form colonies (Supplementary Figure 5B). It corresponded to the proportion of wigKR cells
219 that did not transition to spheres upon L-Ara addition, suggesting that wigKR was essential for
220 the recovery of the spheroplasts, as previously observed for cells treated with cell wall targeting
221 antibiotics. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
222 L-Ara is imported in the cytoplasm and metabolically processed
223 We reasoned that metabolic arrest and spheroplast formation were unlikely to result from the
224 action of L-Ara on the surface of V. cholerae or in its periplasm, in contrast to cell wall targeting
225 antibiotics. V. cholerae lacks a bona fide arabinose import and metabolization pathway.
226 However, the effectiveness of the E. coli PBAD promoter regulation by L-Ara suggested that it
227 was at least passively imported in the cytoplasm of V. cholerae cells. To identify putative
228 factors involved in the response of V. cholerae cells to L-Ara, we screened a random
229 transposition (Tn) library for mutants that were insensitive to L-Ara. To limit false positives,
230 the screening was performed using the MCH1 strain carrying an additional copy of the slmA
231 gene that fully transitions to spheres in the presence of as little as 0.01% (w/v) of L-Ara (Figure
232 1C). After Tn mutagenesis, the libraries were screened on M9-MM plates containing 0.1%
233 (w/v) L-Ara. We then isolated single L-Ara resistant colonies on fresh L-Ara plates and verified
234 their resistance to L-Ara in liquid media. Finally, the morphology of the mutants treated with
235 L-Ara was inspected at the microscope.
236 Of the 11 colonies initially detected in the screening, 9 were confirmed to be L-Ara insensitive.
237 They corresponded to Tn insertions in 6 different genes (Table 3 and Supplementary Figure 6).
238 The suppressor capacity of the genes identified in the screening was confirmed by using the
239 corresponding mutant in an ordered V. cholerae mapped Tn library [23] or, if the strain
240 corresponding to the inactivated gene of interest was missing (i.e. vc0263, vc2689), by direct
241 mutagenesis of the gene in a N16961 wild-type background. Comparison of the growth rates of
242 the identified mutants in the presence or absence of L-Ara further confirmed that they were not
243 affected by L-Ara (Supplementary Figure 7). The study was completed by the investigation of
244 the resistance to L-Ara of a few genes belonging to the same enzymatic pathways of the mutants
245 identified in the screening. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
246 Inactivation of vc1325, vc1327, vc1328, vc1595, vc1596 or vc0263 fully restored growth in
247 presence of L-Ara (Figure 4). VC1325, VC1327 and VC1328 are homologues of E. coli MglB,
248 MglA and MglC, respectively. The three corresponding genes form an operon encoding the
249 components of the ABC galactose transport system: MglB is the periplasmic binding protein,
250 MglA the ATP-binding protein and the integral membrane protein MglC is the permease
251 [24,25]. VC1595 is the homologue of the E. coli GalK galactokinase, the first enzyme in the
252 Leloir pathway of galactose metabolism [26]. VC1596 is the homologue of E. coli GalT, the
253 galactose 1-phosphate uridylyltransferase. VC0263 is a homologue of E. coli WcaJ, the
254 initiating enzyme for colanic acid synthesis [27], which was also described to act as a galactose-
255 1-phosphate transferase in vitro [28]. Taken together, these results suggest that L-Ara is
256 imported in the cytoplasm of V. cholerae by the galactose transporter and processed by the
257 galactose catabolic enzymes. In Sinorhizobium meliloti, the arabinose transporter AraABC has
258 been described to play a role in galactose uptake [29], suggesting a similarity in the activity of
259 the arabinose and galactose transporter.
260 Growth of a vc2095 mutant was very slow, and further decreased in the presence of L-Ara.
261 However, its growth was not completely arrested and spherical cells were not detected. VC2095
262 is the homologue of E. coli Pgm phosphoglucomutase, which suggested that the product
263 resulting from the action of the galactose catabolic enzymes on L-Ara entered the glycolysis
264 pathway. Mutants of vc0374, the homologue of the E. coli glucose 6-phosphate isomerase pgi,
265 grew very slowly and L-Ara addition did not further worsen the growth defect. Microscopic
266 inspection of L-Ara treated cells showed the presence of rare spherical cells. In addition, three
267 independent Tn hits were obtained in VC2689, the homologue of E. coli 6-phosphofructokinase
268 PfkA. L-Ara reduced the growth rate of vc2689 mutants (Supplementary Figure 7). However,
269 microscopic inspection revealed a majority of rod-shaped cells with a few isolated spherical
270 cells, suggesting that L-Ara sensitivity was reduced even though not completely suppressed. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
271 The genes involved in the successive reactions of glycolysis were essential and could not be
272 inactivated, with the exception of vc2670, the putative homologue of E. coli triosephosphate
273 isomerase tpiA, which did not suppress L-Ara induced growth inhibition (Supplementary Figure
274 7). Finally, we found that Tn inactivation of vc0395 and vca0774, the homologues of E. coli
275 UTP-glucose 1-phosphate uridylyltransferase GalU and UDP-glucose 4-epimerase GalE,
276 respectively, which are necessary for supplying UDP-glucose and the interconversion of UDP-
277 galactose as part of the galactose metabolism [30], did not suppress L-Ara induced growth
278 inhibition (Supplementary Figure 7).
279 Taken together, these results suggest the growth arrest of V. cholerae cells treated with L-Ara
280 and their transition to spheroplasts is due to the entrance of L-Ara in the galactose and
281 glycolysis pathways.
282 Discussion
283 It was recently reported that L-Ara inhibits the proliferation of V. cholerae [14]. Here, we show
284 that it is associated with a change in the morphology of the cell from a curved rod shape to a
285 spherical form (Figure 1). The spherical cells lose the ability to divide and stop growing in
286 volume over time, suggesting a major metabolic arrest (Figure 2). We found that L-Ara induced
287 spherical cells are spheroplasts, i.e. they are cell wall deficient (Table 2 and Supplementary
288 Figure 4). Nevertheless, they remain viable and once L-Ara is removed from the environment
289 they resume proliferation and revert to the original cell shape after a few divisions (Figure 3).
290 Metabolism arrest is linked to the processing of L-Ara by the galactose pathway
291 V. cholerae is known to be sensitive to high concentrations of several carbon sources, including
292 glucose, in starvation conditions [31]. In contrast, L-Ara can stop V. cholerae proliferation in
293 both fast and slow growth conditions (Figure 1). The phenomenon was strictly limited to L-Ara
294 (Table 1). V. cholerae lacks a bona fide L-Ara import and degradation pathway. However, the
295 effectiveness of the regulation of the E. coli PBAD promoter in V. cholerae indicated that it was bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
296 at least passively imported in the cytoplasm of the bacterium, where it interfered with the
297 metabolism. We performed a Tn-based screening to determine which cellular processes might
298 be involved in the action of L-Ara. The majority of the genes identified belonged to the
299 galactose Leloir and glycolysis pathways, starting from the uptake of L-Ara through the ABC
300 galactose transport system (Table 3). The distribution of suppressors all along the galactose
301 catabolic pathway and the first steps of glycolysis strongly supports the hypothesis that L-Ara
302 is mistakenly recognized as a substrate of the galactose Leloir pathway and that through a series
303 of enzymatic reactions it is converted into a product that enters the glycolysis pathway (Figure
304 4). The last enzyme in the glycolytic pathway we identified in the Tn suppressor screening is
305 the putative homologue of 6-phosphofructokinase. These results suggest that L-Ara is
306 converted into a phosphorylated sugar by-product that cannot be further metabolized. We found
307 that two enzymes can perform the last step of the Leloir pathway on L-Ara, the Galactose 1-
308 phosphate uridylyltransferase (VC1596) and the product of vc0263. Inactivation of either
309 vc1596 or vc0263 was sufficient to make V. cholerae insensitive to L-Ara, further suggesting
310 that growth arrest is not directly triggered by the non-metabolizable phosphorylated sugar by-
311 product of L-Ara but by the metabolic imbalance created by its accumulation. Correspondingly,
312 L-Ara arrests growth all the more efficiently in slow growing conditions, i.e. in conditions
313 where the concentration of L-Ara is high compared to other nutrients or in cells carrying
314 mutations that already affect their physiological state (Figure 1). Several other studies suggested
315 that accumulation of a phosphate ester metabolite could perturb growth: L-Ara inhibits the
316 growth of E. coli araD mutants because of the accumulation of L-ribulose 5-phosphate [32];
317 Galactose inhibits the growth of E. coli galT mutants because of the accumulation of galactose
318 1-phosphate [33,34]; Rhamnose stops the growth of Salmonella Typhi strains defective in the
319 rhamnose degradation pathway because of the accumulation of L-rhamnulose 1-phosphate
320 [32,35]. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
321 L-Ara mediated metabolic perturbation does not prevent the use of PBAD
322 Importantly, the realization that L-Ara can perturb the metabolism of V. cholerae does not
323 jeopardize previous results obtained with the E. coli PBAD expression system in this bacterium
324 since L-Ara concentrations lower than those that promote growth arrest and spheroplasts
325 formation were almost always used (Figure 1). However, our study indicates that special care
326 should be taken in future works when using the E. coli PBAD expression system in mutants of V.
327 cholerae (Figure 1). According to our results, two methods can be proposed to avoid metabolic
328 artefacts when using the PBAD expression system in V. cholerae. On one hand, cells can be
329 grown in media containing glucose, which represses expression of the galactose import and
330 degradation pathway [34]. On the other hand, experiments can be performed in cells that can
331 import L-Ara but are insensitive to it by mutating the galK gene, which codes for the first
332 enzyme that processes L-Ara in the cytoplasm (Figure 4).
333 Spheroplasts formation results from an imbalance in the cell wall maintenance enzymes
334 It was reported that V. cholerae cells tolerate exposure to antibiotics inhibiting cell wall
335 synthesis by transitioning to non-dividing cell wall deficient spherical cells [5]. Like L-Ara
336 induced spheroplasts, those spherical cells are osmo-resistant and can revert to proliferating
337 rods after removal of the antibiotics [5]. However, in contrast to L-Ara induced spheroplasts,
338 V. cholerae cells exposed to antibiotics inhibiting cell wall synthesis grow in volume over time,
339 suggesting that they are not dormant (Figure 3, [36,37]). In this regard, antibiotic treated cells
340 are more similar to bacterial L-forms, which can be obtained in osmotic stabilizing media in
341 several microorganisms, including E. coli, by treating cells with lysozyme [38], by adding the
342 β-lactam cefsulodin (a specific inhibitor of the penicillin binding proteins PBP1A and PBP1B)
343 [39,40], or by inhibiting synthesis of the Lipid II cell wall precursor with fosfomycin [41].
344 The series of steps and the proteins involved in the formation of antibiotic induced spherical
345 cells in V. cholerae have been partially elucidated [5]. The PG amidase AmiB is responsible bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
346 for the first breach in the cell wall from which blebs originate, although other enzymes can
347 initiate the process in its absence. For the transition from blebs to viable spheres either the D,D-
348 endopeptidase ShyA or ShyC are required, otherwise cells lyse after blebbing. At last, lytic
349 transglycosylases appear to play a role in cell wall degradation and PG turnover and recycling.
350 The analysis of the muropeptide composition of L-Ara treated V. cholerae cells showed that
351 they still maintained a residual amount of PG whose structure was remarkably similar to that
352 described for E. coli cefsulodin-induced L-forms [39]. The dramatic decrease in the average
353 chain-length and corresponding increase in anhydro-muropeptides hint to a higher activity of
354 lytic transglycosylases in cleaving the PG and producing shorter chains. The increase in DAP-
355 DAP cross-linkage, an unusual kind of cross-linkage specifically generated by L,D-
356 transpeptidases [42], further suggests that the metabolic arrest induced by L-Ara inhibits PBP
357 enzymes and stimulates the L,D-transpeptidase activity (LdtA [43]). Taken together, these
358 results suggest that L-Ara promotes the formation of spheroplasts because it induces a
359 metabolic arrest that leads to an imbalance between PG synthesis and degradation. In this
360 regard, L-Ara treatment mimics the action of cell wall targeting enzymes. However, mid-cell
361 does not appear to be the preferential site for bleb formation in L-Ara treated cells as described
362 for cells exposed to antibiotics targeting cell wall synthesis. Therefore AmiB, which is
363 supposedly active only at the septal site [44,45], might not be the preferred initiator of cell wall
364 cleavage in the presence of L-Ara.
365 Proliferation restart results from the restoration of a normal metabolism
366 wigKR spheroplasts increased in volume when L-Ara was removed from the growth media,
367 demonstrating that cell metabolism was very rapidly restored (Figure 3D). However, WigKR
368 played an essential role in the recovery of L-Ara treated cells: the expansion in volume of the
369 wigKR spherical cells upon L-Ara removal led to their lysis (Figure 3D). These results indicate
370 that the recovery of a constitutive level of PG synthesis was not sufficient to expand the residual bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
371 amount of cell wall left in the spheroplasts to accommodate the increase in cellular material, as
372 it was observed for the spheroplasts induced by cell wall targeting antibiotics. Taken together,
373 these results fit with the idea that the histidine kinase/response regulator pair wigKR serves to
374 induce a higher expression of the full set of cell wall synthetic genes in response to cell wall
375 damages [22].
376 Spheroplast formation could be a general survival strategy of Vibrios
377 A multitude of Vibrio species, including V. vulnificus, V. shiloi, V. tasmaniensis, V.
378 parahaemolyticus and V. cholerae form Viable But Not Culturable (VBNC) cells in cold
379 temperatures [46–48]. Cold-induced VBNC cells are osmo-resistant and remain viable for
380 months until the next warm season, which partly explains the seasonality of Vibrio epidemics
381 [46–48]. Cold is supposed to perturb the metabolic activities of the above-mentioned species,
382 plunging them in a state of so-called dormancy [49–51]. L-Ara induced spheroplasts are akin
383 to cold-induced VBNC cells: their formation results from the perturbation of their metabolic
384 activities by a by-product of L-Ara degradation (Figure 4); they are apparently dormant since
385 they do not grow in volume (Figure 3); they are osmo-resistant and return to proliferation upon
386 the removal of L-Ara (Figure 3). Vibrios have also been described to persist as coccoidal bodies
387 in biofilms in the natural aquatic environment [4,52–54]. Interestingly, L-Ara was found to
388 induce biofilm formation in Vibrio fischeri, even though no remarks were made about cell
389 morphology [55]. As in V. cholerae spheroplasts, mutations in GalK or the galactose transporter
390 suppressed the phenotypic action of L-Ara [55]. Taken together, these observations suggest that
391 the effect of L-Ara on V. cholerae could serve as a paradigm for a general survival strategy of
392 Vibrio species under a variety of environmental stresses. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
393 Methods
394 Plasmids and strains
395 Bacterial strains and plasmids used in this study are listed in Supplementary Table 1. Strains
396 were rendered competent by the insertion of hapR by specific transposition and constructed by
397 natural transformation. Engineered strains were confirmed by PCR.
398 Growth curves
399 Cells were grown at 30°C in M9 minimal medium supplemented with 0.2% (w/v) fructose and
400 1 μg/ml thiamine (M9-MM), M9-MM + 0.1% casamino acids (M9-MM + CAA) and Luria-
401 Bertani broth (LB) in a 96-well microtitre plate and the optical density at 600 nm followed over
402 time in a Tecan plate reader. The growth curves plotted are the average of three replicates; the
403 standard deviation is represented for each time point. For CFU and rod to sphere kinetics, cells
404 were grown in flasks in M9-MM at 30°C, 0.2% (w/v) L-Ara was added when indicated.
405 Samples were taken every hour for plating and/or microscopic inspection. Three replicates were
406 performed for each experiment.
407 L-Ara survival assay
408 Over-night wild-type (EPV50) and wigKR deletion mutant (EGV515) cultures were diluted 200
409 times in M9-MM, followed by 2 hours of growth at 30°C before 0.2% (w/v) L-Ara was added.
410 Cells were checked for transition to spherical morphology at the microscope. Serial dilutions
411 of T0 (before L-Ara addition) and T10 (after L-Ara treatment) samples were plated on LB plates
412 and the number of colonies used to calculate the CFU at T0 and T10. The ratio CFU T10 / CFU
413 T0 is used to calculate the percentage of cells able to survive L-Ara treatment and revert to
414 proliferation.
415 Microscopy
416 Cells were spread on a 1% (w/v) agar pad (ultrapure agarose, Invitrogen) for analysis. For
417 snapshots, images were acquired using a DM6000-B (Leica) microscope. For time-lapse bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
418 analyses the agarose pad was made using M9-MM with 0.2% (w/v) L-Ara if needed and images
419 were acquired using an Evolve 512 EMCCD camera (Roper Scientific) attached to an Axio
420 Observe spinning disk (Zeiss). To observe rod to sphere transition on agarose pads, 0.2% (w/v)
421 L-Ara was added in liquid M9-MM cultures 2 hours before transferring cells on agarose pads
422 containing L-Ara and starting microscopic imaging.
423 Transposon mutagenesis screen
424 The transposon mutagenesis was performed conjugating the E. coli strain SM10 λ pir/pSC189,
425 which carries a mini-Himar transposon associated to a kanamycin resistance, with the V.
426 cholerae strain EGV299. We constructed two Tn libraries of approximately 300,000 clones
427 each. Tn mutants were selected on M9-MM plates containing kanamycin and 0.1% (w/v) L-
428 Ara incubated overnight at 30°C. Mutants able to grow on plate were isolated and inspected at
429 the microscope for growth and morphology in presence of L-Ara. An arbitrary PCR followed
430 by DNA sequencing was performed to determine the transposon insertion site.
431 Peptidoglycan analysis
432 EGV217 over-night cultures were diluted 200 times in M9-MM and grown at 30°C. 100 ml
433 were centrifuged after 7 hours of growth and 1L of culture, to which 0.2% (w/v) L-Ara was
434 added after 2 hours, was harvested after further 7 hours of incubation. Cells were checked for
435 complete transition to spherical morphology at the microscope before harvesting. Previously
436 described methods were followed for muropeptide isolation and ultra-performance liquid
437 chromatography (UPLC) analysis [56,57]. After boiling for 2 hours cell pellets with SDS
438 (sodium dodecyl sulfate) the lysates were left stirring over night at room temperature. Cell wall
439 material was pelleted, washed with MQ water to remove the SDS, and digested with pronase E
440 to remove Braun’s lipoprotein. Purified peptidoglycan was re-suspended in MQ water and
441 treated over night with muramidase at 37°C. Soluble muropeptides were reduced with sodium
442 borohydride and the pH then adjusted to 3.5 with phosphoric acid. Samples were injected in an bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
443 UPLC system to obtain the muropeptide profiles. UPLC separation was performed on a Waters
444 UPLC system equipped with an ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm
445 × 150 mm (Waters) and a dual wavelength absorbance detector using a linear gradient from
446 buffer A (phosphate buffer 50 mM, pH 4.35) to buffer B (phosphate buffer 50 mM, pH 4.95,
447 methanol 15% (v/v)) in a 28-min run with a 0.25 mL/min flow. Elution of muropeptides was
448 detected at 204 nm. Identity of the peaks was assigned by comparison of the retention times
449 and profiles to other chromatograms in which mass spectrometry data have been collected. The
450 relative amounts of the muropeptides and the percentage of cross-linkage were calculated as
451 described by Glauner et al. [19]. To estimate the amount of peptidoglycan per cell, the total
452 area of the chromatogram was normalized to the OD of the culture. All values are the means of
453 three independent experiments.
454
455
456 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
457 Figures
A L-Ara
0% 0.02% 0.1% 0.2% 0.5% 1%
M
M
-
9
M
A
A
C
+
M
M
-
9
M
B L
B Cold Amp C MCH1 PBAD::slmA N16961 PBAD::ssb
a
r
1
A
6
-
9
L
6
1
%
N
1
0 . 458 0
459 Figure 1. L-Ara induces loss of rod shape. A. Phase contrast images of V. cholerae N16961
460 cells grown at 30°C in the indicated media with increasing concentrations of L-Ara. B. Phase
461 contrast images of V. cholerae N16961 cells incubated in M9-MM at 4°C for 6 weeks (left
462 panel) and grown in LB with 100 μM Ampicillin (Amp) at 30°C (right panel). C. Phase contrast
463 images of V. cholerae EGV299 (MCH1 PBAD::YGFP-slmA) and EGV300 (N16961 PBAD::ssb-
464 YGFP) cells grown in M9-MM at 30°C in presence of 0.01% (w/v) L-Ara. Scale bars = 2 μm.
465
466
467
468
469
470
471
472 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
473
474 Figure 2. Transition dynamics to spherical cells. Cells were grown in M9-MM at 30°C. L-
475 Ara was added at a concentration of 0.2% (w/v) when indicated. A. Kinetics of V. cholerae
476 morphological change from rods to spherical cells. Cell shape was inspected at the microscope
477 every hour after L-Ara addition. A representative image for each cell category (rod,
478 transitioning, spherical) is represented. Mean of three independent replicates and the standard
479 deviation are represented. B. Diameter distribution of V. cholerae spherical cells treated with
480 L-Ara for 10 hours. C. Average diameter of V. cholerae spherical cells over time. Mean of three
481 independent replicates and the standard deviation are represented. D. Transition from rods to
482 spherical forms. N16961 cells were mounted on a M9-MM agarose pad containing 0.2% (w/v)
483 L-Ara. Bright-field still images from time-lapse microscopy experiments. Images were taken
484 every 5 minutes for 12.5 hours. Scale bars = 2 μm. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B Not treated L-Ara 100 1000 90
6 80
0 1
) 70
x
1
100 %
-
l
( 60
l
m a
50
v
i
U
v F
r 40 C
10 u
S
g 30 o l + L-Ara 20 1 10 0 1 2 3 4 5 6 7 0 Time (h) N16961
C 0’ 225’ 280’ 340’ 390’ 415’
440’ 465’ 500’ 525’ 560’ 650’
D 0’ 120’ 170’ 250’ 350’ 450’
475’ 535’ 540’ 665’ 785’ 790’
485
486 Figure 3. Recovery of growth and rod shape. Cells were grown in M9-MM at 30°C. L-Ara
487 was added at a concentration of 0.2% (w/v) when indicated. Viable colony count (CFU) of V.
488 cholerae cells grown with and without L-Ara over time (A) and after 10 hours (B). Mean of
489 three independent replicates and the standard deviation are represented. In the time-lapse
490 experiments, cells were grown in M9-MM + 0.2% (w/v) L-Ara until they became spherical and
491 then mounted on a M9-MM agarose pad in the absence of L-Ara. Bright-field still images were
492 taken every 5 minutes for 14 hours. C. N16961 cells recovery from spherical CWD forms to
493 rods. D. Spherical wigKR cells are not able to recover rod shape. Scale bars = 2 μm.
494
495 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
D-galactose ABC transporter: VC1325 (1 hit), VC1327 (1 hit), VC1328 (1 hit)
D-galactose D-glucose 1-phosphate UTP-Glucose Galactokinase 1-phosphate VC1595 (1 hit) uridylyltransferase VC0395 D-galactose 1-phosphate UDP-D-glucose
Galactose 1-phosphate uridylyltransferase UDP-Glucose VC1596 4-epimerase OR VCA0774 VC0263 (2 hits)
D-glucose 1-phosphate UDP-D-galactose
Phosphoglucomutase VC2095
D-glucose 6-phosphate
Glucose 6-phosphate isomerase VC0374 D-fructose 6-phosphate
Fructose-1,6 6-Phosphofructokinase bisphosphatase VC2689 (3 hits) VC2544
Fructose-1,6-bisphosphate Fructose-bisphosphate aldolase VC0478
D-glyceraldehyde Dihydroxyacetone 3-phosphate phosphate Triosephosphate isomerase VC2670 496
497 Figure 4. L-Ara insensitive mutants. Schematic representation of the enzymatic pathways
498 targeted by the suppressor screening. In red are the genes that when inactivated suppress the L-
499 Ara induced phenotype (number of hits obtained in the screening in between parentheses), in
500 green the non-suppressors, in blue the gene that could not be inactivated because essential and
501 in black the gene not tested. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
502 Table legends
503
504 Carbon source Growth Spherical cells D-Arabinose + − 505 L-Arabinose − + L-Rhamnose + − 506 Glucose + − Galactose + − 507 Glycerol + − Sucrose + − 508
509 Table 1. Carbon sources tested for N16961 rod shape loss. They were added to M9-MM at a
510 concentration of 0.2% (w/v), with the exception of glycerol at 10 % (v/v).
511
512
513
514
515
516
517
518
519
520
521
522
523 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
524 Muropeptide group Non-treated L-Ara-treated 525 Monomers (%) 48.1 (±0.6) 40.2 (±3.2) 526 Dimers (%) 43.1 (±1.9) 47.2 (±3.1) Trimers (%) 8.8 (±2.5) 12.6 (±4.8) 527 Anhydro-muropeptides (%) 14.8 (±2.3) 21.9 (±3.7) DAP-DAP cross-linked muropeptides (%) 4.9 (±0.7) 7.5 (±0.9) 528 Peptidoglycan feature Non-treated L-Ara-treated Total cross-linkage (%) 37.7 (±1.4) 47.2 (±5.0) 529 Average glycan chain length 11.7 (±1.4) 7.5 (±1.1)
530 Table 2. Quantification of muropeptides, peptidoglycan cross-linking levels and average chain
531 length of L-Ara treated and non-treated cells. Values are the means of three independent
532 experiments and the standard deviation is represented.
533
534
Gene Putative function N. hits vc0263 Galactosyl-transferase 2 vc1325 Galactoside ABC transporter, periplasmic 1 D-galactose/D-glucose binding protein vc1327 Galactoside ABC transporter, ATP-binding 1 protein vc1328 Galactoside ABC transporter, permease 1 protein vc1595 Galactokinase 1 vc2689 6-Phosphofructokinase, isozyme I 3 535
536 Table 3. Suppressor mutants of L-Ara induced toxicity identified in a Tn-based screen.
537
538
539
540
541
542
543
544 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
545 Supplementary Figure legends
546
1.0 M9-MM
0.8
0 0.6
0
6
D O 0.4
0.2
0.0 0 1 2 3 4 5 6 7 8 9 10 Time (h) 0.8 M9-MM + CAA
0.6
0
0 6
D 0.4 O
0.2
0.0 0 1 2 3 4 5 6 7 8 9 10 Time (h) 1.5 LB
1.2
0.9
0
0
6 D
O 0.6
0.3
0.0 0 1 2 3 4 5 6 7 8 9 10 Time (h) No L-Ara L-Ara 0.02% L-Ara 0.1% L-Ara 0.2% L-Ara 0.5% L-Ara 1% 547
548 Supplementary Figure 1. V. cholerae N16961 growth profiles in M9-MM (top panel), M9-
549 MM + CAA (middle panel) and LB (bottom panel) in the presence of increasing concentrations
550 of L-Ara. The optical density at 600 nm over time is the mean of three independent replicates;
551 the standard deviation is represented for each time point.
552 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1.0 N16961
0.8 M9-MM
+ D-Arabinose
0.6 + L-Arabinose
0 0
6 + L-Rhamnose D
O + Glucose 0.4 + Galactose
+ Glycerol
0.2 + Sucrose
0.0 0 1 2 3 4 5 6 7 8 9 10 553 Time (h)
554 Supplementary Figure 2. Carbon sources tested for inhibition of cell growth. V. cholerae
555 N16961 growth profiles in M9-MM in the presence of different carbon sources at a
556 concentration of 0.2% (w/v), with the exception of glycerol at 10 % (v/v). The optical density
557 at 600 nm over time is the mean of three independent replicates; the standard deviation is
558 represented for each time point.
559
560
561
562
563
564
565 Supplementary Figure 3. Localization of the bleb formation. N16961 cells were grown in M9-
566 MM + 0.2% (w/v) L-Ara at 30°C. Phase contrast images. Scale bar = 2 μm.
567 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A
0.2
m
n
4 0
2 0.1 Non-treated
s
b A 0 2 7 12 17 22 27 Retention time (min)
0.02
m
n
4 0.01
0
2
s L-Ara-treated
b A
0 2 7 12 17 22 27 Retention time (min)
B
1.0
l
l
e c
0.8
r
e
p
t 0.6
n
u o
m 0.4
a
G
P 0.2
0.0 Non-treated L-Ara-treated 568
569 Supplementary Figure 4. A. UPLC muropeptides separation. A representative muropeptide
570 profile is presented for cells grown in M9-MM supplemented (red) or not (black) with 0.2%
571 (w/v) L-Ara. B. Peptidoglycan quantification in non-treated and L-Ara treated cells. Amount
572 of PG per cell normalized to the amount obtained for the non-treated cultures. All values are
573 the means of three independent experiments. Mean of three independent replicates and the
574 standard deviation are represented.
575 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
576
577 Supplementary Figure 5. A. Morphological transition from rods to spheres of wild-type
578 N16961 and wigKR mutant cells after addition of 0.2% (w/v) L-Ara. Cells were grown in M9-
579 MM at 30°C and cell shape was inspected at the microscope every hour. A representative image
580 for each cell category (rod, transitioning, spherical) is represented. Scale bar = 2 μm. B. Survival
581 of wild-type N16961 and wigKR mutant cells after incubation in M9-MM + 0.2% (w/v) L-Ara
582 for 10 hours. Data are averages of three biological replicates; the standard deviation is
583 represented.
584
585
586
587
588
589
590 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
2821159 2821432
VC0264 VC0263 VC0262 VC0260
VC0261
1677898 1679559 1680235
VC1328 VC1327 VC1325
VC1326 VC1324
1381872
VC1596 VC1595 VC1594 VC1593
232373 232553 232997
VC2690 VC2689 VC2688 591 VC2691 VC2687
592 Supplementary Figure 6. Maps of the regions of the chromosomes showing the location of
593 Tn insertions in L-Ara insensitive mutants. The arrows represent the sites of insertion.
594
595
596
597 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
0.5 0.6 M9-MM M9-MM + 0.2% L-Ara
0.4
0.4
0.3
0
0
0
0
6
6
D
D
O O 0.2 0.2
0.1
0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (h) Time (h)
WT VC1595 VC1596 VC1325 VC1327 VC1328
0.6 0.6 M9-MM M9-MM + 0.2% L-Ara
0.4 0.4
0
0
0
0
6
6
D
D
O O
0.2 0.2
0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (h) Time (h)
WT VCA0774 VC0395 VC2670 VC0263
0.5 0.25 M9-MM M9-MM + 0.2% L-Ara
0.4 0.20
0.3 0.15
0
0
0
0
6
6
D
D
O O 0.2 0.10
0.1 0.05
0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (h) Time (h) 598 WT VC0374 VC2095 VC2689
599 Supplementary Figure 7. Growth profiles in M9-MM with and without 0.2% (w/v) L-Ara of
600 V. cholerae strains carrying Tn inactivated mutants of genes belonging to the galactose Leloir
601 and glycolysis pathways. The optical density at 600 nm over time is the mean of three
602 independent replicates; the standard deviation is represented for each time point.
603
604
605 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
606 Acknowledgements
607 We would like to acknowledge financial support from the Agence Nationale pour la Recherche
608 [ANR19-CE35-0013-01 SurVi]. We thank C. Possoz for helpful discussions and Y. Yamaichi
609 for providing the ordered V. cholerae mapped Tn library. Research in the Cava laboratory is
610 supported by the Laboratory of Molecular Infection Medicine Sweden (MIMS), the Swedish
611 Research Council (VR), the Knut and Alice Wallenberg Foundation (KAW) and the Kempe
612 Foundation. S.B.H. was supported by a Martin Escudero Postdoctoral fellowship.
613
614 References
615 1. The Biology of Vibrios. American Society of Microbiology; 2006.
616 doi:10.1128/9781555815714
617 2. Chaiyanan S, Chaiyanan S, Grim C, Maugel T, Huq A, Colwell RR. Ultrastructure of coccoid
618 viable but non-culturable Vibrio cholerae. Environmental Microbiology. 2007;9: 393–
619 402. doi:10.1111/j.1462-2920.2006.01150.x
620 3. Huq A, Colwell RR, Rahman R, Ali A, Chowdhury MA, Parveen S, et al. Detection of Vibrio
621 cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture
622 methods. Appl Environ Microbiol. 1990;56: 2370–2373.
623 4. Alam M, Sultana M, Nair GB, Siddique AK, Hasan NA, Sack RB, et al. Viable but
624 nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in
625 cholera transmission. Proc Natl Acad Sci USA. 2007;104: 17801–17806.
626 doi:10.1073/pnas.0705599104
627 5. Dörr T, Davis BM, Waldor MK. Endopeptidase-mediated beta lactam tolerance. PLoS
628 Pathog. 2015;11: e1004850. doi:10.1371/journal.ppat.1004850 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
629 6. Clemens JD, Nair GB, Ahmed T, Qadri F, Holmgren J. Cholera. Lancet. 2017;390: 1539–
630 1549. doi:10.1016/S0140-6736(17)30559-7
631 7. Kim EJ, Lee D, Moon SH, Lee CH, Kim SJ, Lee JH, et al. Molecular Insights Into the
632 Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants. PLoS Pathog.
633 2014;10: e1004384. doi:10.1371/journal.ppat.1004384
634 8. Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY, Haley BJ, et al. Comparative genomics reveals
635 mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae.
636 Proc Natl Acad Sci U S A. 2009;106: 15442–7. doi:0907787106 [pii]
637 10.1073/pnas.0907787106
638 9. Weill F-X, Domman D, Njamkepo E, Tarr C, Rauzier J, Fawal N, et al. Genomic history of
639 the seventh pandemic of cholera in Africa. Science. 2017;358: 785–789.
640 doi:10.1126/science.aad5901
641 10. Domman D, Quilici M-L, Dorman MJ, Njamkepo E, Mutreja A, Mather AE, et al. Integrated
642 view of Vibrio cholerae in the Americas. Science. 2017;358: 789–793.
643 doi:10.1126/science.aao2136
644 11. Mutreja A, Kim DW, Thomson NR, Connor TR, Lee JH, Kariuki S, et al. Evidence for several
645 waves of global transmission in the seventh cholera pandemic. Nature. 2011;477: 462–
646 465. doi:10.1038/nature10392
647 12. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level
648 expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177:
649 4121–30. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
650 13. Lee N, Francklyn C, Hamilton EP. Arabinose-induced binding of AraC protein to araI2
651 activates the araBAD operon promoter. Proc Natl Acad Sci USA. 1987;84: 8814–8818.
652 14. Golder T, Mukhopadhyay AK, Koley H, Nandy RK. Nonmetabolizable arabinose inhibits
653 Vibrio cholerae growth in M9 medium with gluconate as sole carbon source. Jpn J Infect
654 Dis. 2020. doi:10.7883/yoken.JJID.2019.304
655 15. Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria.
656 FEMS Microbiol Rev. 2010;34: 415–425. doi:10.1111/j.1574-6976.2009.00200.x
657 16. Bianco PR, Lyubchenko YL. SSB and the RecG DNA helicase: an intimate association to
658 rescue a stalled replication fork. Protein Sci. 2017;26: 638–649. doi:10.1002/pro.3114
659 17. Galli E, Poidevin M, Le Bars R, Desfontaines J-M, Muresan L, Paly E, et al. Cell division
660 licensing in the multi-chromosomal Vibrio cholerae bacterium. Nature Microbiology.
661 2016;1: 16094.
662 18. Allan EJ, Hoischen C, Gumpert J. Bacterial L-forms. Adv Appl Microbiol. 2009;68: 1–39.
663 doi:10.1016/S0065-2164(09)01201-5
664 19. Glauner B, Höltje JV, Schwarz U. The composition of the murein of Escherichia coli. J Biol
665 Chem. 1988;263: 10088–10095.
666 20. van Heijenoort J. Peptidoglycan hydrolases of Escherichia coli. Microbiol Mol Biol Rev.
667 2011;75: 636–663. doi:10.1128/MMBR.00022-11
668 21. Vollmer W, Joris B, Charlier P, Foster S. Bacterial peptidoglycan (murein) hydrolases.
669 FEMS Microbiol Rev. 2008;32: 259–286. doi:10.1111/j.1574-6976.2007.00099.x bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
670 22. Dörr T, Alvarez L, Delgado F, Davis BM, Cava F, Waldor MK. A cell wall damage response
671 mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance.
672 Proc Natl Acad Sci USA. 2016;113: 404–409. doi:10.1073/pnas.1520333113
673 23. Cameron DE, Urbach JM, Mekalanos JJ. A defined transposon mutant library and its use
674 in identifying motility genes in Vibrio cholerae. Proc Natl Acad Sci USA. 2008;105: 8736–
675 8741. doi:10.1073/pnas.0803281105
676 24. Harayama S, Bollinger J, Iino T, Hazelbauer GL. Characterization of the mgl operon of
677 Escherichia coli by transposon mutagenesis and molecular cloning. J Bacteriol. 1983;153:
678 408–415.
679 25. Hogg RW, Voelker C, Von Carlowitz I. Nucleotide sequence and analysis of the mgl operon
680 of Escherichia coli K12. Mol Gen Genet. 1991;229: 453–459.
681 26. Kalckar HM, Kurahashi K, Jordan E. Hereditary defects in galactose metabolism in
682 Escherichia coli mutants I. determination of enzyme activities. Proc Natl Acad Sci USA.
683 1959;45: 1776–1786.
684 27. Stevenson G, Andrianopoulos K, Hobbs M, Reeves PR. Organization of the Escherichia coli
685 K-12 gene cluster responsible for production of the extracellular polysaccharide colanic
686 acid. J Bacteriol. 1996;178: 4885–4893. doi:10.1128/jb.178.16.4885-4893.1996
687 28. Patel KB, Toh E, Fernandez XB, Hanuszkiewicz A, Hardy GG, Brun YV, et al. Functional
688 characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate
689 transferases of Escherichia coli and Caulobacter crescentus. J Bacteriol. 2012;194: 2646–
690 2657. doi:10.1128/JB.06052-11 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
691 29. Geddes BA, Oresnik IJ. Inability to catabolize galactose leads to increased ability to
692 compete for nodule occupancy in Sinorhizobium meliloti. J Bacteriol. 2012;194: 5044–
693 5053. doi:10.1128/JB.00982-12
694 30. Frey PA. The Leloir pathway: a mechanistic imperative for three enzymes to change the
695 stereochemical configuration of a single carbon in galactose. FASEB J. 1996;10: 461–470.
696 31. Shiba T, Hill RT, Straube WL, Colwell RR. Decrease in culturability of Vibrio cholerae
697 caused by glucose. Appl Environ Microbiol. 1995;61: 2583–2588.
698 32. Englesberg E, Anderson RL, Weinberg R, Lee N, Hoffee P, Huttenhauer G, et al. L-
699 Arabinose-sensitive, L-ribulose 5-phosphate 4-epimerase-deficient mutants of
700 Escherichia coli. J Bacteriol. 1962;84: 137–146.
701 33. Kurahashi K, Wahba AJ. Interference with growth of certain Escherichia coli mutants by
702 galactose. Biochim Biophys Acta. 1958;30: 298–302.
703 34. Yarmolinsky MB, Wiesmeyer H, Kalckar HM, Jordan E. Hereditary defects in galactose
704 metabolism in Escherichia coli mutants II. Galactose-induced sensitivity. Proc Natl Acad
705 Sci USA. 1959;45: 1786–1791.
706 35. Englesberg E, Baron LS. Mutation to L-rhamnose resistance and transduction to L-
707 rhamnose utilization in Salmonella typhosa. J Bacteriol. 1959;78: 675–686.
708 36. Weaver AI, Murphy SG, Umans BD, Tallavajhala S, Onyekwere I, Wittels S, et al. Genetic
709 Determinants of Penicillin Tolerance in Vibrio cholerae. Antimicrob Agents Chemother.
710 2018;62. doi:10.1128/AAC.01326-18 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
711 37. Cross T, Ransegnola B, Shin J-H, Weaver A, Fauntleroy K, VanNieuwenhze MS, et al.
712 Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens. Antimicrob
713 Agents Chemother. 2019;63. doi:10.1128/AAC.00756-19
714 38. Ranjit DK, Young KD. The Rcs stress response and accessory envelope proteins are
715 required for de novo generation of cell shape in Escherichia coli. J Bacteriol. 2013;195:
716 2452–2462. doi:10.1128/JB.00160-13
717 39. Joseleau-Petit D, Liébart J-C, Ayala JA, D’Ari R. Unstable Escherichia coli L forms revisited:
718 growth requires peptidoglycan synthesis. J Bacteriol. 2007;189: 6512–6520.
719 doi:10.1128/JB.00273-07
720 40. Cambré A, Zimmermann M, Sauer U, Vivijs B, Cenens W, Michiels CW, et al. Metabolite
721 profiling and peptidoglycan analysis of transient cell wall-deficient bacteria in a new
722 Escherichia coli model system. Environ Microbiol. 2015;17: 1586–1599.
723 doi:10.1111/1462-2920.12594
724 41. Mercier R, Kawai Y, Errington J. General principles for the formation and proliferation of
725 a wall-free (L-form) state in bacteria. Elife. 2014;3. doi:10.7554/eLife.04629
726 42. Magnet S, Dubost L, Marie A, Arthur M, Gutmann L. Identification of the L,D-
727 transpeptidases for peptidoglycan cross-linking in Escherichia coli. J Bacteriol. 2008;190:
728 4782–4785. doi:10.1128/JB.00025-08
729 43. Cava F, de Pedro MA, Lam H, Davis BM, Waldor MK. Distinct pathways for modification
730 of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 2011;30: 3442–3453.
731 doi:10.1038/emboj.2011.246 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
732 44. Peters NT, Dinh T, Bernhardt TG. A fail-safe mechanism in the septal ring assembly
733 pathway generated by the sequential recruitment of cell separation amidases and their
734 activators. J Bacteriol. 2011;193: 4973–4983. doi:10.1128/JB.00316-11
735 45. Möll A, Dörr T, Alvarez L, Chao MC, Davis BM, Cava F, et al. Cell separation in Vibrio
736 cholerae is mediated by a single amidase whose action is modulated by two
737 nonredundant activators. J Bacteriol. 2014;196: 3937–3948. doi:10.1128/JB.02094-14
738 46. Alam M, Sultana M, Nair GB, Sack RB, Sack DA, Siddique AK, et al. Toxigenic Vibrio
739 cholerae in the aquatic environment of Mathbaria, Bangladesh. Appl Environ Microbiol.
740 2006;72: 2849–2855. doi:10.1128/AEM.72.4.2849-2855.2006
741 47. Huq A, Colwell RR, Rahman R, Ali A, Chowdhury MA, Parveen S, et al. Detection of Vibrio
742 cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture
743 methods. Appl Environ Microbiol. 1990;56: 2370–2373. Available:
744 https://aem.asm.org/content/56/8/2370
745 48. Su C-P, Jane W-N, Wong H. Changes of ultrastructure and stress tolerance of Vibrio
746 parahaemolyticus upon entering viable but nonculturable state. Int J Food Microbiol.
747 2013;160: 360–366. doi:10.1016/j.ijfoodmicro.2012.11.012
748 49. Rittershaus ESC, Baek S-H, Sassetti CM. The normalcy of dormancy: common themes in
749 microbial quiescence. Cell Host Microbe. 2013;13: 643–651.
750 doi:10.1016/j.chom.2013.05.012
751 50. Lewis K. Persister cells. Annu Rev Microbiol. 2010;64: 357–372.
752 doi:10.1146/annurev.micro.112408.134306 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.203711; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
753 51. Hayes CS, Low DA. Signals of growth regulation in bacteria. Curr Opin Microbiol. 2009;12:
754 667–673. doi:10.1016/j.mib.2009.09.006
755 52. Rodrigues S, Paillard C, Le Pennec G, Dufour A, Bazire A. Vibrio tapetis, the Causative
756 Agent of Brown Ring Disease, Forms Biofilms with Spherical Components. Front
757 Microbiol. 2015;6: 1384. doi:10.3389/fmicb.2015.01384
758 53. Oliver JD, Hite F, McDougald D, Andon NL, Simpson LM. Entry into, and resuscitation
759 from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.
760 Appl Environ Microbiol. 1995;61: 2624–2630.
761 54. Grimes DJ, Atwell RW, Brayton PR, Palmer LM, Rollins DM, Roszak DB, et al. The fate of
762 enteric pathogenic bacteria in estuarine and marine environments. Microbiol Sci. 1986;3:
763 324–329.
764 55. Visick KL, Quirke KP, McEwen SM. Arabinose induces pellicle formation by Vibrio fischeri.
765 Appl Environ Microbiol. 2013;79: 2069–2080. doi:10.1128/AEM.03526-12
766 56. Desmarais SM, De Pedro MA, Cava F, Huang KC. Peptidoglycan at its peaks: how
767 chromatographic analyses can reveal bacterial cell wall structure and assembly. Mol
768 Microbiol. 2013;89: 1–13. doi:10.1111/mmi.12266
769 57. Möll A, Dörr T, Alvarez L, Davis BM, Cava F, Waldor MK. A D, D-carboxypeptidase is
770 required for Vibrio cholerae halotolerance. Environ Microbiol. 2015;17: 527–540.
771 doi:10.1111/1462-2920.12779
772