Content uploaded by Alexander Kros
Author content
All content in this area was uploaded by Alexander Kros on Sep 09, 2021
Content may be subject to copyright.
Generating heterokaryotic cells via bacterial cell-cell fusion
1
2
Shraddha Shitut1,2,3, Meng-Jie Shen2, Bart Claushuis3, Rico J. E. Derks4, Martin
3
Giera4, Daniel Rozen3, Dennis Claessen3, Alexander Kros2
4
5
1 Origins Centre, Groningen, the Netherlands
6
2 Dept. Supramolecular & Biomaterials chemistry, Leiden Institute of Chemistry, Leiden
7
University, the Netherlands
8
3 Institute of Biology, Leiden University, the Netherlands
9
4 Center for Proteomics and Metabolomics, Leiden University Medical Center, the
10
Netherlands
11
12
Correspondence to: s.s.shitut@lic.leidenuniv.nl (SS),
13
d.claessen@biology.leidenuniv.nl (DC), a.kros@chem.leidenuniv.nl (AK)
14
15
Abstract
16
Cell-cell fusion is fundamentally important for tissue repair, virus transmission, and
17
genetic recombination, among other functions. Fusion has been mainly studied in
18
eukaryotic cells and lipid vesicles, while cell-cell fusion in bacteria is less well
19
characterized, due to the cell wall acting as a fusion-limiting barrier. Here we use cell
20
wall-deficient bacteria to investigate the dynamics of cell fusion in bacteria that
21
replicate without their cell wall. Stable, replicating cells containing differently labeled
22
chromosomes were successfully obtained from fusion. We find that the rate of cell-cell
23
fusion depends on the fluidity of cell membranes. Furthermore, we show that not only
24
the efficiency but also the specificity of cell-cell fusion can be controlled via a pair of
25
synthetic membrane-associated lipopeptides. Our results provide a molecular handle
26
to understand and control cell-cell fusion to generate heterokaryotic cells, which was
27
an important step in the evolution of protocells and of increasing importance for the
28
design of synthetic cells.
29
30
31
32
33
34
35
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Introduction
36
The structural and functional complexity of modern bacterial cells evolved gradually
37
over hundreds of millions of years from much simpler enclosed protocells (Szostak,
38
Bartel, and Luisi 2001). These early cells are thought to have resembled self-
39
organizing lipid spheres containing stable catalytic actitivity or primitive metabolism
40
(Monnard and Deamer 2002), but lacking a rigid cell wall. Lipid vesicles are widely
41
used to study the behavior of protocells because they are capable of
42
compartmentalization as well as growth and proliferation (Szostak, Bartel, and Luisi
43
2001; Adamala and Luisi 2011). Proliferation of such vesicles involves dramatic shape
44
perturbations, such as fission, tubulation, and vesiclulation, which likely preceded the
45
coordinated cell division of modern walled bacteria (Svetina 2009; Hanczyc and
46
Szostak 2004). However, because lipid vesicles are inherently limited in terms of their
47
internal cytoplasmic complexity, consisting of only minimal catalytic components, new
48
models are needed that more closely resemble protocells to effectively study their
49
early evolution (Briers et al. 2012; Errington et al. 2016). This is particularly needed to
50
examine mechanisms and genetic consequences of cell fusion, an early mechanism
51
of microbial horizontal gene transfer (Kotnik 2013; Soucy, Huang, and Gogarten 2015;
52
Naor and Gophna 2013).
53
Cell fusion has been studied in many different eukaryotic cell types (Chen et al.
54
2007) and is crucial for tissue repair and regeneration, phenotypic diversity, viral
55
transmission and recombination (Ogle, Cascalho, and Platt 2005). The process of
56
fusion proceeds via several steps: cell adhesion, recognition of cell surface
57
components, membrane remodelling and in some cases nuclear fusion (Zito et al.
58
2016). These processes are highly influenced by lipid-lipid interactions
59
(Chernomordik, Kozlov, and Zimmerberg 1995) which have been studied using coarse
60
grained lipid models and lipid vesicles (Smeijers et al. 2006; Marrink and Mark 2003).
61
Fusion in eukaryotic cells is induced via SNARE proteins that form complexes to
62
bridge together membranes by pulling cells close to each other (Hanson, Heuser, and
63
Jahn 1997). The potential for SNARE proteins, or related tools that bridge membranes,
64
to facilitate bacterial fusion have not yet been explored. Studying cell/membrane
65
fusion in eukaryotes and lipid vesicles have unravelled details of the molecular
66
mechanism of membrane fusion; however these systems are highly divergent in terms
67
of cellular and molecular complexity and are not representative of bacterial fusion,
68
which may be common in species lacking a cell wall.
69
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Many bacterial species can transiently shed their cell wall when exposed to
70
environmental stressors like cell wall targeting antibiotics and osmotic stress
71
(Claessen and Errington 2019). When these stressors are removed, wall-deficient
72
cells can rebuild their cell wall and revert to their walled state. Alternatively, prolonged
73
exposure to these stressors can lead to the formation of so-called L-forms, which can
74
efficiently propagate without their wall (Mercier, Kawai, and Errington 2014; Innes and
75
Allan 2001; Glover, Yang, and Zhang 2009; Studer et al. 2016). Much like lipid
76
vesicles, L-form growth and division is regulated by physicochemical forces that
77
deform the cell membrane, leading to an irregular assortment of progeny cells.
78
However, L-forms contain the sophisticated machinery of modern cells which is
79
lacking in protocell models based on giant lipid vesicles (Briers et al. 2012). This
80
makes them suitable to understanding the dynamics and consequences of cellular
81
fusion, as well as to identify factors that affect this process.
82
In this study we show that fusion between L-form cells is a dynamic process
83
whose frequency is dependent on the age of the bacterial culture; this, in turn, is
84
determined by the fluidity of the cell membrane, which we confirm by chemically
85
manipulating membrane fluidity. In addition, we demonstrate for the first time that
86
complementary lipidated coiled coil lipopeptides (structurally similar to SNARE
87
proteins) increase the efficiency and specificity of cell-cell fusion. Importantly, fusants
88
resulting from this process are viable and express markers from both parental
89
chromosomes. This opens up avenues to design complex heterokaryotic/hybrid cells
90
that have potential not only to answer questions on evolution of complexity but also
91
enable novel applications in biotechnology.
92
93
Results
94
A dual marker system for identifying cell-cell fusion
95
In order to study cell-cell fusion, we created two fluorescent strains by integrating
96
plasmids pGreen or pRed2 into the attB site in the genome of an L-form derivative of
97
the actinobacterium K. viridifaciens (Fig. 1A). The strain carrying pGreen constitutively
98
expresses EGFP and is apramycin resistant, while the strain carrying pRed2
99
constitutively expresses mCherry and is hygromycin resistant (Fig. 1A). We first
100
confirmed resistance to these antibiotics by determining the susceptibility of each
101
strain to both antibiotics (Fig. 1B, supplementary fig. 1A). The strain expressing
102
resistance to apramycin (referred to as AG [for Apramycin-Green]) was able to grow
103
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
at 50 µg mL-1 apramycin. The strain that was hygromycin resistant (referred to as HR
104
[for Hygromycin-Red]) could grow at 100 µg mL-1 hygromycin. Resistance to one
105
antibiotic did not provide cross-resistance to the other. Confirmation of the
106
fluorescence reporters was obtained via microscopy with cytoplasmic eGFP detected
107
in the AG strain and mCherry detected in the HR strain (Fig. 1C).
108
109
Figure 1. L-forms used in the study. (A) The wildtype Kitasatospora viridifaciens delta L-form strain
110
was genetically modified to either express apramycin resistance and green fluorescence (AG) or
111
hygromycin resistance and red fluorescence (HR). Each reporter pair (antibiotic resistance+
112
fluorescence gene) was introduced via a plasmid using the ɸC31 integration system. (B) Antibiotic
113
susceptibility testing showed growth of the desired strain at 50 µg/ml apramycin for AG and 100 µg/ml
114
hygromycin for HR. (C) Visual confirmation of fluorescence reporters using microscopy indicated a
115
positive signal in the green channel for AG and in the red channel for HR. Scale bar represents 10 µm.
116
117
Fusion of L-form using centrifugation and PEG
118
L-forms show structural resemblance to protoplasts that are often used for genome
119
reshuffling in plants and bacteria via the process of cell-cell fusion. After fusion these
120
protoplasts can revert back to their walled state. To analyse the ability of L-forms to
121
fuse, we tested some commonly used methods for protoplast fusion (Kieser et al.
122
2000; Baltz and Matsushima 1981; Gokhale, Puntambekar, and Deobagkar 1993)
123
namely, mechanical force induced fusion via centrifugation and PEG-mediated fusion
124
(Fig. 2). Non-specific fusion between AG and HR strains via centrifugation or PEG
125
could result in three different genotypes: AG/HR, AG/AG and HR/HR. However,
126
genetically identical fusants (AG/AG and HR/HR) would not grow on selection plates
127
containing both antibiotics (supplementary fig. 1B). Fusion frequencies determined by
128
growth on both antibiotics are therefore an underestimate of true fusion rates.
129
Centrifuging mixtures of AG and HR at 500 xg resulted in the highest fusion efficiency
130
(1.5 in 105 cells); however, the pellet formed in this case was difficult to handle.
131
Increasing centrifugation to 1000 xg reduced the fusion efficiency to less than 1 fused
132
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
cell per 105 cells, and no fusion was observed at speeds above 6000 xg due to cell
133
lysis (Fig. 3A). The fusion efficiency in the presence of PEG was highest at 10 w%
134
PEG with 1 fused cell per 105 cells (Fig. 3B). Higher PEG concentrations, such as 50
135
w% that is commonly used for protoplast fusion, caused dramatic cell lysis, suggesting
136
that the membrane composition of L-forms is different from protoplasts.
137
138
139
140
To verify that the cells growing on plates with both antibiotics (supplementary fig. 1B)
141
were true fusants, we used microscopy. A small patch of biomass growing on media
142
with both antibiotics was imaged using fluorescence microscopy (Fig. 3C). The
143
percent of pixels that were double labelled (i.e. containing both green and red
144
emission) was higher for cells that had undergone fusion via PEG (21.52%) compared
145
to centrifugal force (11.92%). These patches of double labelled cells indicate the
146
presence and subsequent expression of both sets of marker (AG and HR). The
147
presence of green and red patches in the colonies can be attributed to the fact that
148
the polyploid L-forms may consist of an unequal ratio of the two chromosome types.
149
An unequal ratio and expression of markers can lead to a predominantly green (more
150
AG than HR) or red (more HR than AG) colony appearance. Taken together these
151
results show that cell-cell fusion of L-forms is possible and that the resulting colonies
152
contain both chromosomes.
153
154
155
156
Figure 2. Schematic of L-form fusion.
Fusion was obtained by two types of methods:
non-specific (centrifugation,
poly(ethyleneglycol)-PEG) and cell-specific
(coiled coil lipopeptides). The process of
fusion (black box) and the outcome (grey box)
differs in both cases. For non-specific fusion
the membranes come together by dehydration
induced by PEG or physical centrifugal force.
In the case of coiled coil lipopeptides (CPE
and CPK), they dock in the membrane using
the cholesterol anchor and pull together
opposing membranes upon complementary
coiling. This complementarity results in fusion
of only oppositely labelled cells unlike that in
the non-specific methods.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
157
Figure 3. Cell-cell fusion of L-forms. Non specific cell fusion was carried out using either a physical
158
method (centrifugation (A)) or chemical method (poly(ethyleneglycol) (B)). The fusion efficiency was
159
calculated by dividing the total cell count obtained on double selection media with the cell count of
160
individual parent strain (AG or HR). Increasing centrifugal force leads to a decrease in efficiency (one-
161
way ANOVA, f=15, p=9.77x10-9, groupwise comparison Tukey’s HSD). Poly(ethyleneglycol)
162
concentrations also affected fusion efficiency (one-way ANOVA, f=22, p=0.033, groupwise comparison
163
Tukey’s HSD) with 10 %w resulting in the highest efficiency of fusion. (C) Fluorescence microscopy of
164
colonies on double antibiotic media after fusion via centrifugation (top panel) and PEG 10 %w (bottom
165
panel). Fluorescence expression (EGFP and mCherry) is indicated as percent in the top right corner of
166
each image and was calculated using ImageJ/Fiji. The overlay image (third column) shows the percent
167
or area occupied by both green and red pixels and is slightly higher for PEG induced fusion. Scale bar
168
= 100 µm.
169
170
171
172
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Fused cells are viable and can proliferate
173
Successful cell-cell fusion events between different L-form strains combines the
174
cytoplasmic contents and genomes of these cells. To study whether these fused cells
175
(i.e. fusant) are viable, timelapse microscopy of individual cells was performed. In
176
viable growing L-forms, membrane extension and blebbing takes place first along with
177
deformation of cell shape (Mercier, Kawai, and Errington 2013; Studer et al. 2016).
178
This is followed by daughter cell formation which tend to remain attached to the mother
179
cell. Given the non-binary nature of cell division in wall deficient cells it was difficult to
180
track the exact number of daughter cells originating from one mother cell. Using the
181
wildtype L-forms as a reference for cell growth we looked for the same pattern in fused
182
cells which were viable in the presence of both antibiotics. Colonies from a fusion
183
event were inoculated in double selection liquid media to obtain suspended cultures
184
that could be introduced into a 96 well plate for timelapse imaging in an automated
185
microscope. We applied brightfield and fluorescence imaging every 10 min for over a
186
period of 16 hours (Fig. 4). Importantly, the fused L-forms follow the growth
187
characteristics of wild-type/parental strains as evidenced by blebbing and membrane
188
deformation, as well as smaller daughter cells visibly attached to mother cells (Fig. 4,
189
supplementary movie 1). The fusants also show growth upon subculture into fresh
190
medium containing both selection pressures (supplementary Fig. 2).
191
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
192
Figure 4. Viability of fused cells. Growth and division of fused cell was tracked over time with
193
brightfield (BF) and fluorescence (GFP and mCherry) microscopy. Images were taken every 10 minutes
194
for a total of 16 hours. The panels (top-BF, middle-GFP, bottom-mCherry) consist of a select few images
195
over this time period (labelled on the top left corner in minutes). White arrows indicate growing cells
196
and membrane extensions. Fused cells also express both fluorescence markers made possible due to
197
cell-cell fusion.
198
199
Membrane fluidity influences fusion efficiency.
200
The bacterial cell membrane largely consists of (phospho)lipids and fatty acids,
201
together with other minor components. The characteristics of these lipids and fatty
202
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
acids (FA), such as the degree of unsaturation and headgroup composition, determine
203
the physical properties of a membrane. The fluidity of membranes is an important
204
factor governing its fission and fusion ability (Mercier, Domínguez-Cuevas, and
205
Errington 2012; Prives and Shinitzky 1977). Membrane fluidity of L-form cells was
206
quantified as generalized polarization (GP) using the Laurdan dye assay (Scheinpflug,
207
Krylova, and Strahl 2017). This GP value can range from -1 to +1 and inversely
208
correlates to membrane fluidity (i.e., a low GP value indicates a more fluid membrane).
209
Measuring the fluidity for L-forms grown for 1, 3, 5 and 7 days, resulted in a significant
210
GP value increase over time (Fig. 5A, rho=0.732, p=1.87x10-6), indicating that
211
membrane rigidity increases as the cultures age. Importantly, this change in fluidity
212
with culture age negatively correlated with the fusion efficiency, as younger cultures
213
fused at twice the efficiency of older cultures (Fig. 5A inset, unpaired t test, p=2.22x10-
214
6). To assess the underlying molecular causes for this shift in fluidity, the membrane
215
lipid and FA composition was analyzed using mass spectrometry from L-form cultures
216
of different ages. Over a 7-day period, there was a significant shift in (phospho)lipid/FA
217
composition as the fraction of saturated FAs increased at the expense of unsaturated
218
FAs (Fig. 5B, top panel). This change is consistent with previous reports in
219
Streptomyces sp. and Bacillus sp. showing that membrane fluidity decreases due to
220
the presence of saturated FAs that stack tightly and thereby make membranes rigid
221
(Mercier, Domínguez-Cuevas, and Errington 2012; Hoischen et al. 1997). In addition,
222
the percent of phosphatidylethanolamine (PE) which is known to affect membrane
223
curvature declines with culture age in L-forms. Both factors, an increase in saturated
224
FAs and a decrease in PE, likely underlie the shift in fusion frequency with colony age,
225
although by different mechanisms.
226
To causally confirm the impact of membrane fluidity with fusion efficiency, we
227
directly manipulated membrane fluidity by adding PEG into the medium, which is
228
known to induce fusion between two membranes by hydrogen bonding and force
229
adjacent membranes into close proximity via dehydration (MacDonald 1985;
230
Wojcieszyn et al. 1983). When we tested the effect of increasing PEG concentrations
231
on L-form membrane fluidity, we observed a significant positive correlation between
232
GP values and PEG concentrations (rho=0.834, p=1.41x10-6) (Fig. 5C) This shows
233
that an increase in PEG leads to reduced membrane fluidity in L-forms. In turn, this
234
caused a decrease in fusion efficiency. Thus a high GP value (i.e. low membrane
235
fluidity) results in low fusion (rho=-0.762, p=3.74x10-5) (Fig. 5D).
236
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Taken together these results show that increased membrane fluidity facilitates
237
fusion, which varies naturally during the growth of L-form cells and can be chemically
238
manipulated by the addition of PEG.
239
240
Figure 5. Membrane fluidity affects L-form fusion. (A) Fluidity of L-form membranes was quantified
241
as a generalized polarization (GP) value using the Laurdan dye assay. A strong positive correlation was
242
obtained between GP value and the period of growth indicating a decrease in membrane fluidity with
243
increasing culture age (Spearman’s rank correlation test). Age of the culture also has an effect on fusion
244
efficiency (inset, 2 sample t test, p=2.22x10-6, n=3) with young 2 day old cultures fusing more efficiently
245
than older 7 day old cultures. (B) Analysis of membrane lipids of cultures from different period of growth
246
(1, 3, 5 and 7 day) indicated a change in the percent of saturated and unsaturated fatty acids over time.
247
Specifically the triglyceraldehyde (TG) and phosphatidylethanolamine (PE) show a strong decrease
248
between 1 and 3 day. Both lipids are required for fluidity of the membrane. (C) Positive correlation
249
obtained between GP value and the percent of PEG indicating a decrease in membrane fluidity with
250
increasing concentration of PEG (Spearman’s rank correlation test). (D) The GP value shows a strong
251
negative correlation with fusion efficiency. A low percent of PEG (10%) leads to slightly more fluid
252
membranes compared to a high PEG percent (50%) resulting in higher fusion (Spearman’s rank
253
correlation test). The grayscale (bottom left corner) indicates PEG percent ranging from 10 to 50.
254
255
256
257
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Coiled coil lipopeptides localize to L-form membranes and alter membrane
258
fluidity
259
PEG-mediated fusion and centrifugation cause non-specific cell fusion and this can
260
result in a low percent of fused cells expressing both EGFP and mCherry (Fig. 2). The
261
recent use of lipidated peptides in cell fusion has shown great promise to improve
262
fusion efficiency, with examples of successful fusion between liposomes or liposomes
263
with various eukaryotic cell lines (Rabe et al. 2014; Yang, Shimada, et al. 2016; Kong
264
et al. 2020; Yang, Bahreman, et al. 2016). Coiled coil is a common protein structural
265
motif (supplementary Fig. 3) that contains two or more alpha-helices wrapped around
266
each other to form a left-handed superhelical structure (Koukalová et al. 2018; Robson
267
Marsden and Kros 2010). In previous studies, de novo designed coiled coil forming
268
lipopeptides K4 and E4 were conjugated to cholesterol via a flexible PEG-4 spacer,
269
yielding lipopeptides denoted as CPK4 and CPE4 (Versluis et al. 2013; Zope et al.
270
2013). Using this coiled coil membrane fusion system, efficient liposome-liposome and
271
cell-liposome fusion has been achieved resulting in efficient cytosolic delivery of cargo
272
(Rabe et al. 2014; Yang, Shimada, et al. 2016; Kong et al. 2020). Since L-forms do
273
not posses a cell wall and its outer membrane is structurally similar to (giant) lipid
274
vesicles, we investigated whether coiled-coil lipopeptides CPE4/CPK4 can be applied
275
to increase the L-form fusion efficiency and introduce cell-specificity. First, we tested
276
whether lipopeptide CPK4 could be inserted in the L-form membrane and still form a
277
coiled coil with its binding partner lipopeptide E4 (Fig. 2, supplementary fig. 3).
278
Incorporating the CPK4 lipopeptide in the membrane allowed docking of the
279
complementary fluorescent labeled peptide E4 (fluo-E4; Fig. 6A). Docking was also
280
observed when CPE4 was incorporated in the L-form membrane, followed by the
281
addition of fluorescent labeled peptide fluo-K4. In contrast, no fluorescence was
282
observed when only fluo-K4 or fluo-E4 was added to L-forms (Fig. 6B). Using image
283
analysis software, we further confirmed membrane localization of the lipopeptide-
284
fluorescent dye conjugate by assessing the fluorescence intensity across the cell
285
along a transect line. A combined plot (supplementary fig. 4) of these intensity values
286
across 10 cells indicates coinciding peaks of fluorescence values of the lipopeptide
287
conjugates with that of gray values of the cell membrane (seen as dark grey rings in
288
brightfield images). The fluorescence intensity on L-form membranes was more
289
distinct when CPE4/fluo-K4 was used as compared to CPK4/fluo-E4 (supplementary
290
fig. 4A). Altogether, these results demonstrate for the first time that lipopeptides can
291
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
be readily incorporated into L-form membranes and serve as a docking point for the
292
complementary (lipo)peptides.
293
294
Figure 6. Coiled coil lipopeptides integrate in L-form membranes. (A) Confocal microscopy images
295
(fluorescence (FL) and overlay (FL+DIC)) indicating peptide CPE4 or CPK4 insertion into the L-form
296
membranes and coiled-coil formation with complementary peptides (fluo-K4 or fluo-E4). White arrows
297
indicate clear membrane insertion. (B) In the absence of CPE4 or CPK4 no binding of the complementary
298
fluorescent peptides (fluo-K4 or fluo-E4) was observed. Experiments were performed at 30ºC, L-forms
299
in P-buffer were incubated with 10 µM of CPE4 or CPK4 for 30 minutes. Subsequently the unbound
300
peptide was washed via centrifugation and the complementary fluorescent peptides were added. Scale
301
bar = 5 µM.
302
303
The incorporation of lipopeptides in L-form membranes prompted us to
304
investigate whether they also influenced membrane fluidity. To test this, L-forms
305
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
expressing red fluorescent protein (AR and HR strains) were modified with either non-
306
fluorescent labeled CPE4 and CPK4 so as not to interfere with the emission spectra of
307
the Laurdan dye. The observed GP values reveal that CPK4 and CPE4 affect the
308
fluidity of L-forms differently. While CPE4 decreased fluidity in the AR strain (Fig. 7A),
309
both lipopeptides increased fluidity in the HR strain (Fig. 7B). Interestingly the effect
310
of increased fluidity due to PEG (10 w%) was only observed in the AR strain. These
311
differences in fluidity effects are likely caused by the presence of antibiotics during
312
culturing of the strains prior to the experiment, which are required to avoid
313
contamination in the cultures (supplementary fig. 5). Antibiotics are known to affect
314
membrane fluidity (Bessa, Ferreira, and Gameiro 2018), however the exact
315
mechanism by which they do so is unclear. This inherent difference was observed in
316
the basal GP values of control samples (-0.02 for HR and -0.08 for AR, Fig. 7A-B) as
317
well as in separate measurements for fluidity of strains in the absence and presence
318
of antibiotics (0.01 for HR and -0.10 for AR, supplementary fig. 5). However, all
319
treatments (PEG/lipopeptide) are compared to the control sample of individual strain
320
type; hence, the change in GP value is indeed due to the lipopeptide interaction and
321
the to the presence of antibiotics.
322
We next examined how these changes in fluidity affect the process of
323
lipopeptide-mediated fusion. For this, L-form cultures were first adjusted to the same
324
density and split into aliquots. The aliquots were then either untreated (control), treated
325
with PEG or increasing concentrations of the lipopeptide that previously caused an
326
increase in fluidity. HR strains were hence pretreated with CPE4 and AG strains were
327
treated with CPK4. After treatment for 30 minutes the excess PEG and lipopeptides
328
were removed by centrifugation and the L-forms were resuspended in fresh P-buffer
329
containing DnaseI. The cultures were then thoroughly mixed in a 1:1 ratio, incubated
330
for 30 minutes at 30°C and subsequently plated on selection media for cell
331
quantification. The observed fusion efficiency for each treatment relative to control
332
revealed that treatment of HR with CPE4 and AG with CPK4 results in a high fusion
333
efficiency as compared to 10 w% PEG or the centrifuged control (Fig. 7C).
334
Furthermore, fusion efficiency was not only dependent on lipopeptide concentration
335
(i.e. decreased fusion at 100 μM) but also on the lipopeptide specificity since AG
336
treated with CPE4 resulted in basal level of fusion similar to the control. Higher
337
lipopeptide concentrations also visibly affected cells, causing lysis (data not shown).
338
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Together these results confirm that cell specific fusion of L-forms can be achieved
339
using fusogenic coiled coil lipopeptides.
340
The two approaches (non-specific via PEG and centrifugation and cell-specific
341
using lipopeptides) used here seem to influence fusion by altering membrane fluidity
342
and bringing membranes together. We then investigated whether combining both
343
fusogens would result in an overall higher fusion efficiency. For this the cells were first
344
treated with the lipopeptides (AG L-forms with CPK4 and HR L-forms with CPE4) and
345
split into two aliquots. The first aliquot was directly subjected to fusion by mixing the
346
cultures in a 1:1 ratio whereas the second aliquot was mixed and treated with PEG.
347
Here the PEG remained in the environment during the process of fusion. Efficiency
348
calculations showed a 3-fold higher relative fusion in the latter (Fig. 7D) indicating that
349
combining lipopeptides and PEG is optimal for cell-cell fusion. The presence of
350
lipopeptides on the cell surface aids in complementary L-form pairing (AG with HR)
351
bringing the opposing membranes in close proximity, which is an important first step
352
in fusion. Additionaly PEG potentially further reduces the space by membrane
353
dehydration thus facilitating fusion events. Colony imaging further confirmed the
354
presence of more double labelled cells in treatment with PEG (supplementary Fig. 6).
355
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
356
Figure 7. Coiled coil lipopeptides increase membrane fluidity and cell-specific fusion. (A) The
357
strain AR shows an increased fluidity on treatment with PEG (p=3.06x10-6), a decrease in fluidity on
358
treatment with CPE4 (p=2.13x10-3) and no change in fluidity with CPK4 (One-way ANOVA, F=36,
359
p=4.59x10-18 followed by Tukey’s pairwise comparison) compared to the control (dotted line). (B) The
360
strain HR shows increased fluidity (low GP value) when treated with CPE4 (p=3.11x10-3) and CPK4
361
(p=1.4x10-2) compared to the control (dotted line) whereas no significant change when treated with 10%
362
PEG (One-way ANOVA, F=36, p=2.83x10-18 followed by Tukey’s pairwise comparison). Dotted line is
363
for comparison of GP values to the control where no peptide or PEG was added. (C) The AG and HR
364
strains were individually treated with either PEG, CPE4 or CPK4 at different peptide concentrations to
365
assess the effect on fusion efficiency. Interestingly PEG leads to low fusion despite increasing fluidity
366
because of its non-specific nature. The combination of AG-CPK4 and HR-CPE4 resulted in highest
367
fusion efficiency relative to the basal level. The increase in relative fusion efficiency is concentration
368
dependent as well as peptide dependent (One-way ANOVA, F=30, p=3.47x10-14 followed by Tukey’s
369
pairwise comparison). (D) The AG and HR strains were first treated with either PEG, CPE4 or CPK4.
370
These strains were then directly plated on double selection media in the absence (grey boxes) or
371
presence (black boxes) of 10%w PEG to assess the effect on fusion efficiency. Interestingly PEG leads
372
to low fusion despite increasing fluidity because of its non-specific nature when washed away prior to
373
plating but gives a high efficiency when present during the plating. The treatment with peptides also
374
shows a higher efficiency when in the presence of PEG (Kruskall-Wallis chi-squared = 24.84, p=5.4x10-
375
5, followed by Dunnet’s pairwise comparison) compared to the control where no peptide or PEG was
376
added (dotted line).
377
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Discussion
378
Cell wall deficiency has primarily been studied in the context of stress tolerance and
379
intracellular pathogenicity (Errington et al. 2016). The genetic and metabolic
380
modifications required to survive in this wall-deficient state are also being uncovered
381
which has deepened our understanding of their intriguing biology (Glover, Yang, and
382
Zhang 2009; Kawai et al. 2019). We here show that wall-deficient L-forms are able to
383
fuse with one another and that membrane fluidity is a key factor influencing fusion
384
efficiency. Additionally, we show for the first time targeted fusion between wall-
385
deficient cells using coiled coil lipopeptides. This opens up avenues for application in
386
the field of biotechnology and the design of synthetic cells.
387
L-forms are surrounded by a membrane, which are be sufficiently fluid to allow
388
efficient proliferation. Bacillus subtilis L-forms that have a defect in formation of
389
branched chain fatty acid (BCFA) suffer from decreased membrane fluidity and as a
390
consequence cannot carry out the membrane scission step (Mercier, Domínguez-
391
Cuevas, and Errington 2012). This phenotype was rescued by supplementing the
392
media with BCFAs in the medium. Less is known about the impact of fluidity on
393
bacterial fusion, although older reports on eukaryotic muscle cell cultures suggest that
394
myoblast fusion was preceded by a decrease in membrane viscosity (Prives and
395
Shinitzky 1977). In this work we showed that the membrane fluidity of K. viridifaciens
396
L-forms changes over time. In younger cultures, the fluidity is higher coinciding with
397
the ability of such cells to proliferate efficiently. By contrast, the fluidity decreases in
398
older cultures. The change in fluidity was associated with a change in the ratio of
399
saturated to unsaturated FAs. In our study we found this ratio to be 4.3 for the 1st day
400
of growth which then increased to 11.3 after 3 days (Fig. 5B). Thus the amount of
401
saturated FAs responsible for tighter packing increases over time at the expense of
402
unsaturated FAs. The accumulation of saturated FAs makes the membrane more stiff,
403
which negatively impacts proliferation and fusion efficiency. Notably, compared to
404
protoplasts, L-forms of Streptomyces hygroscopicus contained 6 times more anteiso
405
FAs than protoplasts resulting in more fluid membranes (Hoischen et al. 1997). Our
406
lipidomics analysis also indicates that L-form membrane composition comprised
407
significant amounts of cardiolipin (CL), phosphatidylinositol (PI) and
408
phosphatidylethanolamine (PE). Both CL and PE are fusogenic headgroups shown to
409
induce fusion between liposomes and extracellular vesicles (Driessen et al. 1985), and
410
their the presence may also facilitate L-form fusion.
411
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
A pair of complementary fusogenic coiled coil lipopeptides have been
412
previously developed for the targeted delivery of compounds into eukaryotic cells
413
using liposomes. These eukaryotic-liposome models have also been used extensively
414
to understand the process of cell fusion (Daudey et al. 2017). For the first time we
415
explored targeted fusion with these synthetic lipopeptides between bacterial cells.
416
Interestingly we observed that the lipopeptides readily insert in membranes of L-forms
417
via a cholesterol anchor (Fig. 6). These lipopeptides remained in the membrane even
418
after several washing steps. The lipopeptide segment of CPK4 is known to interact
419
both with its binding partner lipopeptide E4 as well as membranes while the lipopeptide
420
E4 segment of CPE4 does not (Fig. 2). Complementary binding of the lipopeptides
421
brings two opposing membranes in close proximity and ultimately induces fusion
422
(Koukalová et al. 2018; Robson Marsden et al. 2009). The differences in lipopeptide
423
presentation on the surface can explain the complementarity effect on fusion efficiency
424
of L-forms as well (Fig. 7). Given the ease of lipopeptide docking and subsequent
425
stability on the L-forms, coiled coil lipopeptides provide a promising avenue for studies
426
on targeted compound delivery into wall deficient cells. This may be particularly
427
relevant for L-forms associated with recurring urinary tract infections and potentially
428
mycobacterial infections (Mickiewicz et al. 2019; Markova 2017).
429
The costs and benefits of living as a wall deficient cell depends on the
430
environment. Absence of a protective wall makes them sensitive to changes in osmotic
431
pressure and physical agitation. On the other hand, cells without a wall are resistant
432
to a whole class of cell wall targeting antibiotics (penicillins, cephalosporins), transport
433
to the extracellular space is potentially easier and the cells are stably polyploid. These
434
characteristics can make L-forms a unique model system to study not only cell biology
435
but also questions in the fields of biotechnology, evolution and the origin of life (Briers
436
et al. 2012; Errington et al. 2016; Shitut et al. 2020). The process of cell fusion may
437
have been a mechanism of horizontal gene transfer and species diversification in early
438
life (Küppers and Zimmermann 1983). Understanding this process is hence a key
439
aspect of protocell evolution. L-forms are uniquely suited to replicate these processes
440
thereby providing a mechanistic understanding of the causes and consequences of
441
such fusion. First, the use of coiled coil directed fusion can be extended to synthetic
442
cells to obtain fusions that increase cellular complexity. Second, fusion leads to
443
multiple chromosomes in the same cellular compartment which in turn can result in
444
genetic recombination. Such recombination events can then be leveraged to identify
445
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
new microbial products and obtain genomically diverse populations of cells. Finally,
446
cell-cell fusion can also help to understand major transitions on the road to increased
447
organismal complexity like multicellularity and endosymbiosis.
448
449
450
Materials and methods
451
Media and growth conditions
452
All L-form strains were cultured in liquid L phase broth (LPB) and solid L phase media
453
agar (LPMA). LPB consists of a 1:1 mixture of yeast extract malt extract (YEME) and
454
tryptic soy broth supplemented with 10% sucrose (TSBS) and 25 mM MgCl2. LPMA
455
consists of LPB supplemented with 1.5% agar, 5% horse serum and 25 mM MgCl2
456
(Kieser et al. 2000). P-buffer containing sucrose, K2SO4, MgCl2, trace elements,
457
KH2PO4, CaCl2, TES (Kieser et al. 2000) was used for transformation and all fusion
458
experiments supplemented with 1 mg/mL DnaseI (Roche Diangnostics GmbH).
459
Antibiotics apramycin (Duchefa Biochemie) and hygromycin (Duchefa Biochemie)
460
were used for selection and were added at final concentrations of 50 μg/mL and 100
461
μg/mL respectively. Growth conditions for all cultures was 30°C in an orbital shaker
462
(New Brunswick Scientific Innova®) with 100 rpm for the liquid cultures. Centrifugation
463
(Eppendorf Centrifuge 5424) conditions were always 1000 xg for 10 minutes (< 1 mL)
464
or 30 minutes (>10 mL) depending on culture volume. The above mentioned culture
465
conditions and centrifugation settings were applied throughout the study unless
466
mentioned otherwise. All measurements for optical density of samples was done with
467
200 μL culture in a 96 well flat bottom plate (Sarstedt) using the Tecan spectramax
468
platereader.
469
470
Strain and plasmid construction
471
Wall deficient L-form of Kitasatospora viridifaciens was obtained by prolonged
472
exposure to penicillin and lysozyme similar to a previous study (Ramijan et al. 2018).
473
Briefly, 106 spores of Kitasatopsora viridifaciens DSM40239 were grown in 50 mL
474
TSBS media at 30°C and 100 rpm to obtain mycelial biomass. To this biomass 1
475
mg/mL lysozyme (Sigma Aldrich) and 0.6 mg/mL penicillin (Duchefa Biochemie) was
476
added to induce S-cell formation. After 7 days, a dense culture of wall-deficient cells
477
was obtained and subcultured to LPB media containing 6 mg/mL penicillin. This
478
treatment was continued for 5 weeks with subculture into fresh media every week. The
479
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
culture was then tested for growth on LPMA without penicillin and showed only L-form
480
growth. A single colony was picked and inoculated in LPB without penicillin and
481
incubated for 7 days to confirm stability of wall-deficiency and subsequently used for
482
making a culture stock to be stored at -80°C.
483
The strain was further genetically modified to harbour antibiotic resistance
484
genes and fluorescent reporter genes. Two plasmids were used for this purpose
485
namely pGreen (containing the apramycin resistance gene aac(3)IV and a green
486
fluorescent protein reporter gene) and pRed2 (containing the hygromycin resistance
487
gene hph and a red fluorescent reporter gene). Both plasmids contain the Phi C31
488
aatP site and a Phi C31 integrase which allows for integration of the marker set at the
489
attB site in the genome. The pGreen plasmid was obtained from a previous publication
490
where details are provided of the construction (Zacchetti et al. 2016). The pRed2
491
plasmid was constructed by introducing the amplified mCherry gene alongwith a gap1
492
promoter region at the XbaI site in the pIJ82 plasmid. Briefly, the mCherry gene was
493
amplified together with the gap1 promoter using primers (Sigma) mentioned in
494
supplementary table 1 and the pRed plasmid (Zacchetti et al. 2016) as template. The
495
amplified gap1-mCherry product was purified using a kit following instructions of the
496
supplier (Illustra™ GFX™ gel band purification kit). The purified product was
497
introduced into the vector pIJ82 at the XbaI site (New England Biolabs GmbH). This
498
plasmid was first transformed into E. coli DH5alpha for amplification followed by
499
transformation into E. coli ET12567 for demethylation.
500
The plasmids were introduced into the L-forms by polyethylene glycol
501
(PEG1000 NBS Biologicals) induced transformation similar to protoplast
502
transformation with some modifications (Kieser et al. 2000). L-form cultures were
503
grown for 4 days. Cultures were centrifuged to remove the spent media and the pellet
504
was resuspended in 1/4th volume P-buffer. Approximately 500 ng plasmid was added
505
to the resuspended pellet and mixed thoroughly. PEG1000 was added to this mix at a
506
final concentration of 25 w%w and mixed gently. After a brief incubation of 5 minutes
507
on the bench the tube was centrifuged. The supernatant was discarded and the pellet
508
resuspended in LPB medium and incubated for 2 hours. The culture was then
509
centrifuged again and the pellet resuspended in 100 μL LPB for plating on LPMA
510
media containing selective antibiotics apramycin or hygromycin. After 4 days of
511
incubation single colonies were picked and restreaked on LPMA with antibiotics for
512
confirmation along with fluorescence microscopy. The resulting strains were named
513
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
AG for Apramycin-Green and HR for Hygromycin-Red and will be referred so
514
henceforth.
515
To test the antibiotic susceptibility, both strains were grown on LPMA containing
516
with or without either 50 μg/mL apramycin or 100 μg/mL hygromycin for 4 days.
517
Stepwise 10-fold dilution plating was done which allowed for quantifying the number
518
of colonies (CFU/mL).
519
520
L-form fusion
521
Strains AG and HR were grown individually from culture stocks in 20 mL LPB
522
containing the relevant antibiotic. Grown cultures were then centrifuged to remove
523
spent media containing antibiotics and washed with P-buffer twice. The pellet was
524
finally resuspended in 2-3 mL of P-buffer containing DNase I (1 mg/mL) and the
525
density was adjusted to 0.6 OD600. Both strains were then mixed in equal volumes
526
(200 μL) in a fresh microfuge tube and mixed gently followed by incubation at room
527
temperature for 10 minutes. Depending on the treatment, PEG1000 was added at the
528
desired concentration (0 to 50 w%) and mixed by pipetting. For the effect of
529
centrifugation on L-form fusion no PEG was added. After a brief incubation of 5
530
minutes the tubes were centrifuged and the supernatant was discarded. The pellet
531
was resuspended in 100 μL of P-buffer with DNase I and serial dilutions were
532
subsequently plated on LPMA with both antibiotics. Controls were also plated on the
533
same medium such as 100 μL monocultures of each strain to test for cross resistance
534
and 100 μL of 1:1 mix of each strain without fusion (supplementary figure 1). All plates
535
were incubated for 3 days after which colony forming units were calculated to
536
determine the fusion efficiency. Effiiciency was quantified as the CFU/mL on double
537
antibiotic selection media normalized by the CFU/mL of monocultures grown on single
538
antibiotic selection media.
539
540
Microscopy
541
A Zeiss LSM 900 airyscan 2 microscope was used to image the fluorescently labeled
542
strains under 40x magnification. For EGFP an excitation wavelength of 488 nm was
543
used and emission captured at 535 nm whereas for mCherry an excitation wavelength
544
of 535 nm was used and emission captured at 650 nm. Multichannel (fluorescence
545
and brighfield), multi-stack images were captured using the Zen software (Zeiss) and
546
further analyzed using ImageJ/Fiji. Multiple tiles were imaged for colonies to cover a
547
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
large area. These tiles were then stitched and each fluorescence channel was first
548
thresholded to determine the total pixel area. These thresholded images were then
549
used to calculate total area (using the OR function in image calculator) and the fused
550
area (using the AND function). The total area selection was then used to calculate
551
individual pixel area occupied by either green or red pixels and by both.
552
The Lionheart FX automated microscope (BioTek) was used for timelapse
553
imaging of double labeled L-forms after fusion. The fusant strains were precultured in
554
LPB containing both antibiotics for 3 days. These were then centrifuged and
555
resuspended in fresh media with antibiotics and 100 μL of this was added to individual
556
wells in a 96 well black/clear bottom sensoplate (Thermoscientific). The plate was
557
centrifuged for 5 minutes to enable settling of cells. The timelapse imaging was done
558
using a 63x dry objective, set for 3 channels (brightfield, green and red) with imagining
559
every 10 minutes for 16 hours at 30°C. The LED intensity for all channels was 10 and
560
a camera gain of 24. The exposure time was set at the beginning of the imaging
561
according to the reference monoculture strains AG and HR.
562
563
Membrane fluidity assay
564
The membrane fluidity was quantified for cultures of different age and cultures treated
565
with different lipopeptides using the Laurdan dye assay (Scheinpflug, Krylova, and
566
Strahl 2017). All cultures grown in 40 mL volume were first centrifuged followed by
567
resuspension in P-buffer and density adjusted to 0.6 to 0.8 OD600. The cultures were
568
then aliquot according to the treatment for a given biological replicate (i.e. 5 aliquots
569
of 1 mL each for 5 treatments). In case of lipopeptide treatment the lipopeptide was
570
added to the culture at required concentration (5 μM, 10 μM or 100 μM) and all tubes
571
were incubated for 30 minutes at 100 rpm. Centrifugation was carried out to remove
572
excess lipopeptide and the pellet was resuspended in P-buffer. The P-buffer for this
573
assay was always maintained at 30°C so as not to alter fluidity of the membrane. 10
574
mM Laurdan (6-Dodecanoyl-2-Dimethylaminonapthalene, Invitrogen) stock solution
575
was prepared in 100% dimethylformamide (DMF, Sigma) and stored at -20°C in an
576
amber tube to protect from light exposure. This stock solution was used to get a final
577
concentration of 10 μM in the resuspended cultures above. The tubes were inverted
578
to mix the dye sufficiently and then incubated at 30°C for 10 minutes and covered with
579
foil to protect from light exposure. The cultures were then washed 3x in pre-warmed
580
P-buffer containing 1% dimethylsulfoxide (DMSO, Sigma) to ensure removal of
581
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
unbound dye molecules. The final suspension was done in pre-warmed P-buffer and
582
200 μL was transferred to a 96 well black/clear bottom sensoplate (Thermoscientific)
583
for spectroscopy. Fluorescent intensities were measured by excitation at 350 nm and
584
two emission wavelengths (435 and 490 nm). The background values were first
585
subtracted from all sample values followed by estimation of the generalized
586
polarization (GP) value.
587
GP =
I435 – I490
I435 + I490
The GP value ranges from -1 to +1 with low values corresponding to high membrane
588
fluidity.
589
590
Lipid extraction and analysis
591
Cultures of the wildtype L-form were grown for different time periods (1, 3, 5 and 7
592
days). These were centrifuged and resuspended in P-buffer prior to membrane
593
lipidomics. Lipids where extracted using a modified MTBE protocol of Matyash, V. et
594
al. (ref. 10.1194/jlr.D700041-JLR200). In short, 600 μL MTBE and 150 μL methanol
595
were added to the thawed bacteria samples. Samples where briefly vortexed, ultra-
596
sonicated for 10 minutes and shaken at room temperature for 30 minutes. Next, 300
597
µL water was added and the samples where centrifuged for 5 minutes at 18213 ×g at
598
20 ˚C. After centrifugation, the upper layer was collected and transferred to a glass
599
vial. The extraction was repeated by adding 300 µL MTBE and 100 µL methanol.
600
Samples where briefly vortexed and shaken at room temperature for 5 minutes. Next,
601
100 µL water was added and the samples where centrifuged for 5 minutes at 18213
602
×g at 20 ˚C. After centrifugation, the upper layer was collected, and the organic
603
extracts combined. Samples where dried under a gentle stream of nitrogen. After
604
drying samples were reconstituted in 100 µL 2-propanol. After briefly vortexing and
605
ultra-sonication for 5 minutes, 100 µL water was added. Samples were transferred to
606
microvial inserts for analysis
607
Lipidomic analysis of bacteria lipid extracts was performed using a LC-MS/MS
608
based lipid profiling method (PMID: 31972163 DOI: 10.1016/j.bbamem.2020.183200).
609
A Shimadzu Nexera X2 (consisting of two LC30AD pumps, a SIL30AC autosampler,
610
a CTO20AC column oven and a CBM20A controller) (Shimadzu, ‘s Hertogenbosch,
611
The Netherlands) was used to deliver a gradient of water:acetonitrile 80:20 (eluent A)
612
and water:2-propanol:acetonitrile 1:90:9 (eluent B). Both eluents contained 5 mM
613
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
ammonium formate and 0.05% formic acid. The applied gradient, with a column flow
614
of 300 µL/min, was as follows: 0 min 40% B, 10 min 100% B, 12 min 100% B. A
615
Phenomenex Kinetex C18, 2.7 µm particles, 50 × 2.1 mm (Phenomenex, Utrecht, The
616
Netherlands) was used as column with a Phenomenex SecurityGuard Ultra C8, 2.7
617
µm, 5 × 2.1 mm cartridge (Phenomenex, Utrecht, The Netherlands) as guard column.
618
The column was kept at 50 ˚C. The injection volume was 10 µL.
619
The MS was a Sciex TripleTOF 6600 (AB Sciex Netherlands B.V., Nieuwerkerk
620
aan den Ijssel, The Netherlands) operated in positive (ESI+) and negative (ESI-) ESI
621
mode, with the following conditions: ion source gas 1 45 psi, ion source gas 2 50 psi,
622
curtain gas 35 psi, temperature 350˚C, acquisition range m/z 100-1800, ion spray
623
Voltage 5500 V (ESI+) and -4500 V (ESI-), declustering potential 80 V (ESI+) and -80
624
V (ESI-). An information dependent acquisition (IDA) method was used to identify
625
lipids, with the following conditions for MS analysis: collision energy ±10, acquisition
626
time 250 ms and for MS/MS analysis: collision energy ±45, collision energy spread 25,
627
ion release delay 30, ion release width 14, acquisition time 40 ms. The IDA switching
628
criteria were set as follows: for ions greater than m/z 300, which exceed 200 cps,
629
exclude former target for 2 s, exclude isotopes within 1.5 Da, max. candidate ions 20.
630
Before data analysis, raw MS data files were converted with the Reifycs Abf Converter
631
(v1.1) to the Abf file format. MS-DIAL (v4.20), with the FiehnO (VS68) database was
632
used to align the data and identify the different lipids (Tsugawa et al. 2015; 2019;
633
2020). Further processing of the data was done with R version 4.0.2 (R Core Team
634
2014).
635
The relative abundance of specific lipid class vs total relative abundance was
636
used to roughly compare the ratio of each lipid class. The lipids have been sorted into
637
saturated and unsaturated lipids classes. Also, the lipids have been sorted based on
638
head groups (DG, TG, PE, PI) and the ratio of each class have been calculated
639
640
Lipopeptide preparation and treatment
641
Peptide K4 and E4 were synthesized on a CEM Liberty Blue microwave-assisted
642
peptide synthesizer using Fmoc chemistry. 20% piperidine in DMF was used as the
643
deprotection agent. During coupling, DIC was applied as the activator and Oxyma as
644
the base. All peptides were synthesized on a Tentagel S RAM resin (0.22 mmol/g).
645
The resin was swelling for at least 15min before synthesis started. For the coupling, 5
646
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
equivalents of amino acids (2.5 mL in DMF), DIC (1 mL in DMF) and Oxyma (0.5 mL
647
in DMF) were added to the resin in the reaction vessel and were heated to 90 ℃ for 4
648
minutes to facilitate the reaction. For deprotection, 20% of piperidine (4 mL in DMF)
649
was used and heated to 90℃ for 1 minute. Between deprotection and peptide
650
coupling, the resin has been washed three times using DMF. After peptide synthesis,
651
a polyethyleneglycol (PEG)4 linker and cholesterol were coupled manually to the
652
peptide on-resin. 0.1 mmol of each peptide was reacted with 0.2 mmol N3-PEG4-
653
COOH by adding 0.4 mmol HCTU and 0.6 mmol DIPEA in 3 mL DMF. The reaction
654
was performed at room temperature for 5 hours. After thorough washing, 3 mL of 0.5
655
mmol trimethylphosphine in a 1,4-dioxane:H2O (6:1) mixture was added to the resin
656
to reduce the azide group to an amine (overnight reaction). After reduction, the peptide
657
was reacted with cholesteryl hemisuccinate (0.3 mmol) in DMF by adding 0.4 mmol
658
HCTU and 0.6 mmol DIPEA. The reaction was performed at room temperature for 3
659
hours. Lipopeptides were cleaved from the resin using 3 mL of a TFA:triisopropylsilane
660
(97.5:2.5%) mixture and shaking for 50 min. After cleavage, the crude lipopeptides
661
were precipitated by pouring into 45 mL of -20 ℃ diethyl ether:n-hexane (1:1) and
662
isolated by centrifugation. The pellet of the lipopeptides was redissolved by adding 20
663
mL H2O containing 10% acetonitrile and freeze-dried to yield a white powder.
664
Lipopeptides were purified with reversed-phase HPLC on a Shimazu system with two
665
LC-8A pumps and an SPD-20A UV-Vis detector, equipped with a Vydac C4 column
666
(22 mm diameter, 250 mm length, 10 μm particle size). CPK4 was purified using a
667
linear gradient from 20 to 65 % acetonitrile in water (with 0.1% TFA) with a 12 mL/min
668
flow rate over 36 mins. CPE4 was purified using a linear gradient from 20 to 75 %
669
acetonitrile in water (with 0.1% TFA) with a 12 mL/min flow rate over 36 mins. After
670
HPLC purification, all peptides were lyophilized and yielded white powders.
671
For the fluo-K4 and fluo-E4 synthesis, two additional glycine residues were coupled to
672
the N-terminus of the peptides on resin, before the dye was manually coupled by
673
adding 3 mL DMF containing 0.2 mmol 5(6)-carboxyfluorescein, 0.4 mmol HCTU and
674
0.6 mmol DIPEA. The reaction was left at room temperature overnight. The fluo-K4
675
and fluo-E4 were cleaved from the resin using 3 mL of a TFA:triisopropylsilane:H2O
676
(97.5:2.5%) mixture and shaking for 1.5 hours. After cleavage, the crude lipopeptides
677
were precipitated by pouring into 45 mL of -20 ℃ diethyl ether and isolated by
678
centrifugation. The pellet of the lipopeptides was redissolved by adding 20 mL H2O
679
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
containing 10% acetonitrile and freeze-dried to yield a white powder. Fluo-K4 and fluo-
680
E4 were purified using the same HPLC described above equipped with a Kinetix Evo
681
C18 column (21.2 mm diameter, 150 mm length, 5 μm particle size). For the fluo-K4,
682
a linear gradient from 20 to 45% acetonitrile in water (with 0.1% TFA) with a 12 mL/min
683
flow rate over 28 mins was used. For fluo-E4, linear gradient from 20 to 55% was used.
684
After HPLC purification, all peptides were lyophilized and yielded orange powders.
685
The purity of all peptides were determined by LC-MS (supplementary table 2). The
686
structure of all peptides used in this study can be found in supplementary figure 3.
687
Treatment of cultures with different peptides was done by adding externally to cells
688
suspended in P-buffer and incubating for 30 minutes at 30°C 100 rpm. Excess peptide
689
was washed by centrifugation.
690
691
L-form membrane labelling
692
3×108 wild type L-forms were suspended in 1 mL of P-buffer. 10 μL of CPK4 or CPE4
693
(10 mM in DMSO) was added to the L-form suspension to a final concentration of 100
694
μM. After 30 min incubation at 30 ℃ with shaking at 100 rpm, the L-forms were washed
695
two times by centrifugation using P-buffer. The L-forms were then suspended in 900
696
μL P-buffer and 100 μL of fluo-K4 or fluo-E4 (200 μM in P-buffer) was added to a final
697
concentration of 20 μM. After 5min incubation, the L-forms were washed three times
698
using P-buffer to get rid of the free fluorescent lipopeptides. For control experiments,
699
fluo-K4 or fluo-E4 were added to non-lipopeptide modified L-form and incubated for 5
700
min. L-form imaging was performed on a Leica SP8 confocal microscopy. Excitation:
701
488 nm, emission: 500-550 nm.
702
703
Peptide induced L-form fusion
704
Strains AG and HR were grown individually from culture stocks in 20 mL LPB
705
containing the relevant antibiotic. Grown cultures were then centrifuged to remove
706
spent media containing antibiotics and washed with P-buffer twice. The pellet was
707
finally resuspended in 2-3 mL of P-buffer containing DNase I (1 mg mL-1) and the
708
density was adjusted to 0.6 OD600. Peptides were added at required concentrations to
709
1 ml cultutres of individual strains AG and HR. Cultures were then incubated for 30
710
minutes at 30 ℃ with shaking at 100 rpm. Excess and unbound peptide was removed
711
via centrifugation and resuspension of pellet in 1 ml P buffer containing DNase I. Both
712
strains were then mixed in equal volumes (200 μL) in a fresh microfuge tube and mixed
713
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
gently followed by incubation at room temperature for 10 minutes. Depending on the
714
treatment cultures were centrifuged followed by treatment with PEG1000 or simply
715
centrifuged. The pellet was resuspended in 100 μL of P-buffer with DNase I and serial
716
dilutions were subsequently plated on LPMA with both antibiotics. Controls were also
717
plated on the same medium such as 100 μL monocultures of each strain to test for
718
cross resistance and 100 μL of 1:1 mix of each strain without fusion (supplementary
719
figure 1). All plates were incubated for 3 days after which colony forming units were
720
calculated to determine the fusion efficiency. Effiiciency was quantified as the CFU/mL
721
on double antibiotic selection media normalized by the CFU/mL of monocultures
722
grown on single antibiotic selection media.
723
724
Statistical analysis and graphs
725
Statistical analysis of all datasets was done in R version 3.6.1 (R Core Team 2014)
726
using built-in packages. The specific tests performed are mentioned in the results and
727
figure legends. All graphs were produced using the package ggplot2 (Wickham, H.
728
2009).
729
730
Acknowledgements
731
We thank members of the Claessen lab and Kros lab for fruitful discussions and
732
suggestions. S.S. acknowledges the NWA startimpulse (Origins Centre) for funding.
733
734
735
Author contribution
736
S.S., D.C, and A.K. designed the project. S.S. performed all experiments. S.S. and
737
M.S. performed peptide fusion experiments. M.S. prepared all lipopeptides and did
738
microscopy for lipopeptide docking experiments. B.C. prepared the cell-wall-deficient
739
line of K. viridifaciens used in the study. R.D. and M.G. performed the membrane lipid
740
analysis. S.S., D.R., D.C. and A.K. acquired funding. S.S. wrote the first draft followed
741
by revisions from all authors. All authors approved the final manuscript.
742
743
744
745
746
747
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
References
748
Adamala, Katarzyna, and Pier Luigi Luisi. 2011. “Experimental Systems to Explore
749
Life Origin: Perspectives for Understanding Primitive Mechanisms of Cell
750
Division.” Cell Cycle in Development, 1–9.
751
Baltz, Richard H., and Patti Matsushima. 1981. “Protoplast Fusion in Streptomyces:
752
Conditions for Efficient Genetic Recombination and Cell Regeneration.”
753
Microbiology,. Microbiology Society,.
754
Bessa, Lucinda J., Mariana Ferreira, and Paula Gameiro. 2018. “Evaluation of
755
Membrane Fluidity of Multidrug-Resistant Isolates of Escherichia Coli and
756
Staphylococcus Aureus in Presence and Absence of Antibiotics.” Journal of
757
Photochemistry and Photobiology B: Biology 181 (April): 150–56.
758
Briers, Yves, Peter Walde, Markus Schuppler, and Martin J. Loessner. 2012. “How
759
Did Bacterial Ancestors Reproduce? Lessons from L-Form Cells and Giant
760
Lipid Vesicles.” BioEssays 34 (12): 1078–84.
761
Chen, Elizabeth H., Eric Grote, William Mohler, and Agnès Vignery. 2007. “Cell–Cell
762
Fusion.” Membrane Trafficking 581 (11): 2181–93.
763
Chernomordik, L., M. M. Kozlov, and J. Zimmerberg. 1995. “Lipids in Biological
764
Membrane Fusion.” The Journal of Membrane Biology 146 (1): 1–14.
765
Claessen, Dennis, and Jeff Errington. 2019. “Cell Wall Deficiency as a Coping
766
Strategy for Stress.” Trends in Microbiology 27 (12): 1025–33.
767
Daudey, Geert A., Harshal R. Zope, Jens Voskuhl, Alexander Kros, and Aimee L.
768
Boyle. 2017. “Membrane-Fusogen Distance Is Critical for Efficient Coiled-Coil-
769
Peptide-Mediated Liposome Fusion.” Langmuir 33 (43): 12443–52.
770
Driessen, A J, D Hoekstra, G Scherphof, R D Kalicharan, and J Wilschut. 1985. “Low
771
PH-Induced Fusion of Liposomes with Membrane Vesicles Derived from
772
Bacillus Subtilis.” Journal of Biological Chemistry 260 (19): 10880–87.
773
Errington, Jeff, Katarzyna Mickiewicz, Yoshikazu Kawai, and Ling Juan Wu. 2016.
774
“L-Form Bacteria, Chronic Diseases and the Origins of Life.” Philosophical
775
Transactions of the Royal Society B: Biological Sciences 371 (1707):
776
20150494
777
Glover, William A., Yanqin Yang, and Ying Zhang. 2009. “Insights into the Molecular
778
Basis of L-Form Formation and Survival in Escherichia Coli.” PLOS ONE 4
779
(10): e7316.
780
Gokhale, D.V., U.S. Puntambekar, and D.N. Deobagkar. 1993. “Protoplast Fusion: A
781
Tool for Intergeneric Gene Transfer in Bacteria.” Biotechnology Advances 11
782
(2): 199–217.
783
Hanczyc, Martin M, and Jack W Szostak. 2004. “Replicating Vesicles as Models of
784
Primitive Cell Growth and Division.” Current Opinion in Chemical Biology 8
785
(6): 660–64.
786
Hanson, Phyllis I, John E Heuser, and Reinhard Jahn. 1997. “Neurotransmitter
787
Release — Four Years of SNARE Complexes.” Current Opinion in
788
Neurobiology 7 (3): 310–15.
789
Hoischen, C, K Gura, C Luge, and J Gumpert. 1997. “Lipid and Fatty Acid
790
Composition of Cytoplasmic Membranes from Streptomyces Hygroscopicus
791
and Its Stable Protoplast-Type L Form.” Journal of Bacteriology 179 (11):
792
3430.
793
Innes, C.M.J., and E.J. Allan. 2001. “Induction, Growth and Antibiotic Production of
794
Streptomyces Viridifaciens L-Form Bacteria.” Journal of Applied Microbiology
795
90 (3): 301–8.
796
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Kawai, Yoshikazu, Romain Mercier, Katarzyna Mickiewicz, Agnese Serafini, Luiz
797
Pedro Sório de Carvalho, and Jeff Errington. 2019. “Crucial Role for Central
798
Carbon Metabolism in the Bacterial L-Form Switch and Killing by β-Lactam
799
Antibiotics.” Nature Microbiology 4 (10): 1716–26.
800
Kieser, Tobias, Mervyn J. Bibb, Mark J. Buttner, Keith F. Chater, and David A.
801
Hopwood. 2000. Practical Streptomyces Genetics. Vol. 291. John Innes
802
Foundation Norwich.
803
Kong, Li, Quanchi Chen, Frederick Campbell, Ewa Snaar-Jagalska, and Alexander
804
Kros. 2020. “Light-Triggered Cancer Cell Specific Targeting and Liposomal
805
Drug Delivery in a Zebrafish Xenograft Model.” Advanced Healthcare
806
Materials 9 (6): 1901489.
807
Kotnik, Tadej. 2013. “Lightning-Triggered Electroporation and Electrofusion as
808
Possible Contributors to Natural Horizontal Gene Transfer.” Physics of Life
809
Reviews 10 (3): 351–70.
810
Koukalová, Alena, Šárka Pokorná, Aimee L. Boyle, Nestor Lopez Mora, Alexander
811
Kros, Martin Hof, and Radek Šachl. 2018. “Distinct Roles of SNARE-
812
Mimicking Lipopeptides during Initial Steps of Membrane Fusion.” Nanoscale
813
10 (40): 19064–73.
814
Küppers, G., and U. Zimmermann. 1983. “Cell Fusion by Spark Discharge and Its
815
Relevance for Evolutionary Processes.” FEBS Letters 164 (2): 323–29.
816
MacDonald, Ruby I. 1985. “Membrane Fusion Due to Dehydration by Polyethylene
817
Glycol, Dextran, or Sucrose.” Biochemistry 24 (15): 4058–66.
818
Markova, Nadya. 2017. “L-Form Bacteria Cohabitants in Human Blood: Significance
819
for Health and Diseases.” Discovery Medicine 23 (128): 305–13.
820
Marrink, Siewert J., and Alan E. Mark. 2003. “The Mechanism of Vesicle Fusion as
821
Revealed by Molecular Dynamics Simulations.” Journal of the American
822
Chemical Society 125 (37): 11144–45.
823
Mercier, Romain, Patricia Domínguez-Cuevas, and Jeff Errington. 2012. “Crucial
824
Role for Membrane Fluidity in Proliferation of Primitive Cells.” Cell Reports 1
825
(5): 417–23.
826
Mercier, Romain, Yoshikazu Kawai, and Jeff Errington. 2013. “Excess Membrane
827
Synthesis Drives a Primitive Mode of Cell Proliferation.” Cell 152 (5): 997–
828
1007.
829
Mercier, Romain, Yoshikazu Kawai, and Jeff Errington. 2014. “General Principles for
830
the Formation and Proliferation of a Wall-Free (L-Form) State in Bacteria.”
831
ELife 3 (October): e04629.
832
Mickiewicz, Katarzyna M, Yoshikazu Kawai, Lauren Drage, Margarida C Gomes,
833
Frances Davison, Robert Pickard, Judith Hall, Serge Mostowy, Phillip D
834
Aldridge, and Jeff Errington. 2019. “Possible Role of L-Form Switching in
835
Recurrent Urinary Tract Infection.” Nature Communications 10 (1): 4379–
836
4379.
837
Monnard, Pierre-Alain, and David W. Deamer. 2002. “Membrane Self-Assembly
838
Processes: Steps toward the First Cellular Life.” The Anatomical Record 268
839
(3): 196–207.
840
Naor, Adit, and Uri Gophna. 2013. “Cell Fusion and Hybrids in Archaea.”
841
Bioengineered 4 (3): 126–29.
842
Ogle, Brenda M., Marilia Cascalho, and Jeffrey L. Platt. 2005. “Biological
843
Implications of Cell Fusion.” Nature Reviews Molecular Cell Biology 6 (7):
844
567–75.
845
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Prives, J., and M. Shinitzky. 1977. “Increased Membrane Fluidity Precedes Fusion of
846
Muscle Cells.” Nature 268 (5622): 761–63.
847
R Core Team. 2014. R: A Language and Environment for Statistical Computing.
848
Vienna, Austria.: R Foundation for Statistical Computing.
849
Rabe, Martin, Christian Schwieger, Harshal R. Zope, Frank Versluis, and Alexander
850
Kros. 2014. “Membrane Interactions of Fusogenic Coiled-Coil Peptides:
851
Implications for Lipopeptide Mediated Vesicle Fusion.” Langmuir 30 (26):
852
7724–35.
853
Ramijan, Karina, Eveline Ultee, Joost Willemse, Zheren Zhang, Joeri A. J.
854
Wondergem, Anne van der Meij, Doris Heinrich, Ariane Briegel, Gilles P. van
855
Wezel, and Dennis Claessen. 2018. “Stress-Induced Formation of Cell Wall-
856
Deficient Cells in Filamentous Actinomycetes.” Nature Communications 9 (1):
857
5164.
858
Robson Marsden, Hana, Nina A. Elbers, Paul HH Bomans, Nico AJM Sommerdijk,
859
and Alexander Kros. 2009. “A Reduced SNARE Model for Membrane Fusion.”
860
Angewandte Chemie 121 (13): 2366–69.
861
Robson Marsden, Hana, and Alexander Kros. 2010. “Self-Assembly of Coiled Coils
862
in Synthetic Biology: Inspiration and Progress.” Angewandte Chemie
863
International Edition 49 (17): 2988–3005.
864
Scheinpflug, Kathi, Oxana Krylova, and Henrik Strahl. 2017. “Measurement of Cell
865
Membrane Fluidity by Laurdan GP: Fluorescence Spectroscopy and
866
Microscopy.” In Antibiotics: Methods and Protocols, edited by Peter Sass,
867
159–74. New York, NY: Springer New York.
868
Shitut, Shraddha, Güniz Ö. Bergman, Alexander Kros, Daniel E. Rozen, and Dennis
869
Claessen. 2020. “Use of Permanent Wall-Deficient Cells as a System for the
870
Discovery of New-to-Nature Metabolites.” Microorganisms 8 (12).
871
Smeijers, A. F., A. J. Markvoort, K. Pieterse, and P. A. J. Hilbers. 2006. “A Detailed
872
Look at Vesicle Fusion.” The Journal of Physical Chemistry B 110 (26):
873
13212–19.
874
Soucy, Shannon M., Jinling Huang, and Johann Peter Gogarten. 2015. “Horizontal
875
Gene Transfer: Building the Web of Life.” Nature Reviews Genetics 16 (8):
876
472–82.
877
Studer, Patrick, Titu Staubli, Noémi Wieser, Patrick Wolf, Markus Schuppler, and
878
Martin J Loessner. 2016. “Proliferation of Listeria Monocytogenes L-Form
879
Cells by Formation of Internal and External Vesicles.” Nature Communications
880
7 (November): 13631–13631.
881
Svetina, Saša. 2009. “Vesicle Budding and the Origin of Cellular Life.”
882
ChemPhysChem 10 (16): 2769–76.
883
Szostak, Jack W., David P. Bartel, and P. Luigi Luisi. 2001. “Synthesizing Life.”
884
Nature 409 (6818): 387–90.
885
Tsugawa, Hiroshi, Tomas Cajka, Tobias Kind, Yan Ma, Brendan Higgins, Kazutaka
886
Ikeda, Mitsuhiro Kanazawa, Jean VanderGheynst, Oliver Fiehn, and Masanori
887
Arita. 2015. “MS-DIAL: Data-Independent MS/MS Deconvolution for
888
Comprehensive Metabolome Analysis.” Nature Methods 12 (6): 523–26.
889
Tsugawa, Hiroshi, Kazutaka Ikeda, Mikiko Takahashi, Aya Satoh, Yoshifumi Mori,
890
Haruki Uchino, Nobuyuki Okahashi, et al. 2020. “A Lipidome Atlas in MS-DIAL
891
4.” Nature Biotechnology 38 (10): 1159–63.
892
Tsugawa, Hiroshi, Ryo Nakabayashi, Tetsuya Mori, Yutaka Yamada, Mikiko
893
Takahashi, Amit Rai, Ryosuke Sugiyama, et al. 2019. “A Cheminformatics
894
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
Approach to Characterize Metabolomes in Stable-Isotope-Labeled
895
Organisms.” Nature Methods 16 (4): 295–98.
896
Versluis, Frank, Jens Voskuhl, Bartjan van Kolck, Harshal Zope, Marien Bremmer,
897
Tjerk Albregtse, and Alexander Kros. 2013. “In Situ Modification of Plain
898
Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane
899
Fusion.” Journal of the American Chemical Society 135 (21): 8057–62.
900
Wickham, H. 2009. Ggplot2: Elegant Graphics for Data Analysis. Springer New York.
901
Wojcieszyn, J W, R A Schlegel, K Lumley-Sapanski, and K A Jacobson. 1983.
902
“Studies on the Mechanism of Polyethylene Glycol-Mediated Cell Fusion
903
Using Fluorescent Membrane and Cytoplasmic Probes.” Journal of Cell
904
Biology 96 (1): 151–59.
905
Yang, Jian, Azadeh Bahreman, Geert Daudey, Jeroen Bussmann, René C. L.
906
Olsthoorn, and Alexander Kros. 2016. “Drug Delivery via Cell Membrane
907
Fusion Using Lipopeptide Modified Liposomes.” ACS Central Science 2 (9):
908
621–30.
909
Yang, Jian, Yasuhito Shimada, René C. L. Olsthoorn, B. Ewa Snaar-Jagalska,
910
Herman P. Spaink, and Alexander Kros. 2016. “Application of Coiled Coil
911
Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft
912
Model.” ACS Nano 10 (8): 7428–35.
913
Zacchetti, Boris, Joost Willemse, Brand Recter, Dino van Dissel, Gilles P. van
914
Wezel, H. A. B. Wösten, and Dennis Claessen. 2016. “Aggregation of
915
Germlings Is a Major Contributing Factor towards Mycelial Heterogeneity of
916
Streptomyces.” Scientific Reports 6 (1): 27045.
917
Zito, Francesca, Nadia Lampiasi, Igor Kireev, and Roberta Russo. 2016. “United We
918
Stand: Adhesion and Molecular Mechanisms Driving Cell Fusion across
919
Species.” European Journal of Cell Biology 95 (12): 552–62.
920
Zope, Harshal R., Frank Versluis, Anita Ordas, Jens Voskuhl, Herman P. Spaink,
921
and Alexander Kros. 2013. “In Vitro and In Vivo Supramolecular Modification
922
of Biomembranes Using a Lipidated Coiled-Coil Motif.” Angewandte Chemie
923
International Edition 52 (52): 14247–51.
924
925
926
927
928
929
930
931
932
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 1, 2021. ; https://doi.org/10.1101/2021.09.01.458600doi: bioRxiv preprint
