*Fo co espondence:
b amkamp@i am.uni-kiel.de (MB);
d.j.sche e s@ ug.nl (D-JS)
†
These au ho s con ibu ed
equally o his wo k
Compe ing in e es s: The
au ho s decla e ha no
compe ing in e es s exis .
Funding: See page 18
Recei ed: 24 Ma ch 2020
Accep ed: 12 June 2020
Published: 14 July 2020
Re iewing edi o : Taˆm Migno ,
CNRS-Aix Ma seille Uni e si y,
F ance
Copy igh Zielin´ ska e al. This
a icle is dis ibu ed unde he
e ms o he C ea i e Commons
A ibu ion License, which
pe mi s un es ic ed use and
edis ibu ion p o ided ha he
o iginal au ho and sou ce a e
c edi ed.
Flo illin-media ed memb ane luidi y
con ols pep idoglycan syn hesis and
M eB mo emen
Aleksand a Zielin
´ska
1†
, Abigail Sa ie o
2,3†
, Anabela de Sousa Bo ges
1
,
Denis Ma inez
4
, Melanie Be bon
4
, Joe
¨l R Roelo sen
1
, Alwin M Ha man
5,6,7
,
Rinse de Boe
8
, Ida J Van de Klei
8
, Anna KH Hi sch
5,6,7
, Bi gi Habens ein
4
,
Ma c B amkamp
2,3
*, Di k-Jan Sche e s
1
*
1
Molecula Mic obiology, G oningen Biomolecula Sciences and Bio echnology
Ins i u e, Uni e si y o G oningen, G oningen, Ne he lands;
2
Biozen um, Ludwig-
Maximilians-Uni e si a
¨ Mu
¨nchen, Mu
¨nchen, Ge many;
3
Ins i u e o Gene al
Mic obiology, Ch is ian-Alb ech s-Uni e si y, Kiel, Ge many;
4
Ins i u e o Chemis y
& Biology o Memb anes & Nanoobjec s (UMR5248 CBMN), IECB, CNRS, Uni e si e´
Bo deaux, Ins i u Poly echnique Bo deaux, Pessac, F ance;
5
Depa men o D ug
Design and Op imiza ion (DDOP), Helmhol z-Ins i u e o Pha maceu ical Resea ch
Saa land (HIPS) - Helmhol z Cen e o In ec ion Resea ch (HZI), Saa b u
¨cken,
Ge many;
6
Depa men o Pha macy, Saa land Uni e si y, Saa b u
¨cken, Ge many;
7
S a ingh Ins i u e o Chemis y, Uni e si y o G oningen, G oningen, Ne he lands;
8
Molecula Cell Biology, G oningen Biomolecula Sciences and Bio echnology
Ins i u e, Uni e si y o G oningen, G oningen, Ne he lands
Abs ac The bac e ial plasma memb ane is an impo an cellula compa men . In ecen yea s
i has become ob ious ha p o ein complexes and lipids a e no uni o mly dis ibu ed wi hin
memb anes. Cu en hypo heses sugges ha lo illin p o eins a e equi ed o he o ma ion o
complexes o memb ane p o eins including cell-wall syn he ic p o eins. We show he e ha bac e ial
lo illins a e impo an ac o s o memb ane luidi y homeos asis. Loss o lo illins leads o a
dec ease in memb ane luidi y ha in u n leads o al e a ions in M eB dynamics and, as a
consequence, in pep idoglycan syn hesis. These al e a ions a e e e ed when memb ane luidi y is
es o ed by a chemical luidize . In i o, he addi ion o a lo illin inc eases memb ane luidi y o
liposomes. Ou da a suppo a model in which lo illins a e equi ed o di ec con ol o memb ane
luidi y a he han o he o ma ion o p o ein complexes ia di ec p o ein-p o ein in e ac ions.
In oduc ion
The shape o a bac e ium is p edominan ly de ined by he s uc u e o i s pep idoglycan. Al hough
he e is a g ea a ie y in bac e ial shapes, he o e all chemis y o pep idoglycan is e y simila
be ween bac e ia and hus he shape o pep idoglycan is p ima ily de e mined by he empo al and
spa ial egula ion o pep idoglycan syn hesis. In od-shaped bac e ia, pep idoglycan syn hesis is
hough o be media ed by wo p o ein assemblies, he elongasome and he di isome, ha syn he-
sise pep idoglycan along he long axis and ac oss he di ision plane o he cell, espec i ely
(Typas e al., 2012;Zhao e al., 2017). These complexes con ain a se o p o eins equi ed o he
inal s eps o syn hesis and ansloca ion o he pep idoglycan p ecu so , LipidII, om he inne o
he ou e lea le o he cy oplasmic memb ane, and p o eins ha inco po a e LipidII in o pep idogly-
can. These include SEDS (Shape, Elonga ion, Di ision and Spo ula ion) p o eins ha can pe o m
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 1 o 21
RESEARCH ARTICLE
glycosyl ans e ase eac ions (Cho e al., 2016;Meeske e al., 2016;Taguchi e al., 2019), and
Penicillin Binding P o eins (PBPs) ha a e di ided in class A PBPs (aPBPs) ha ca alyse bo h glycosyl
ans e ase and anspep idase eac ions, class B PBPs (bPBPs) ha only ca alyse anspep idase
eac ions and low molecula weigh PBPs ha modi y pep idoglycan, as well as hyd olases
(Zhao e al., 2017;Mo ales Angeles and Sche e s, 2017).
Coo dina ion o hese complexes is linked o cy oskele al elemen s, M eB (-like p o eins) o he
elongasome and F sZ o he di isome. In models, he cy oplasmic memb ane is o en depic ed as a
passi e en i onmen in which hese machine ies a e embedded. Howe e , i is becoming clea ha
he s uc u e o he memb ane plays a c i ical ole in he coo dina ion o pep idoglycan syn hesis
(S ahl and E ing on, 2017). Inwa d memb ane cu a u e se es as a localisa ion igge o M eB
and he elongasome, and enhanced local syn hesis a bulges s aigh ens ou he memb ane su i-
cien o con e sphe ical cells o a od shape (Hussain e al., 2018;U sell e al., 2014). In Bacillus
sub ilis, he mo ion o M eB along he memb ane is associa ed wi h elongasome ac i i y (Domı´-
nguez-Escoba e al., 2011;Ga ne e al., 2011), and he eloci y o M eB pa ches is ela ed o
g ow h a e (Billaudeau e al., 2017), indica ing ha M eB mo ion can be used as a ma ke o elon-
gasome ac i i y. In e es ingly, M eB localises o and o ganises egions o inc eased memb ane luid-
i y (RIF) (S ahl e al., 2014), which in u n is linked o he p esence o LipidII, which a ou s a mo e
luid memb ane and p omo es local memb ane diso de (Ganche e al., 2006;Wi zke e al.,
2016). Inhibi ion o LipidII syn hesis by gene ic o chemical means esul s in a dissolu ion o mem-
b ane s uc u es obse ed wi h he dye FM 4–64 and elease o M eB om he memb ane (Domı´-
nguez-Escoba e al., 2011;Ga ne e al., 2011;Mucho a
´e al., 2011;Schi ne e al., 2015).
Nex o RIFs, memb ane egions o dec eased luidi y ha e been iden i ied in bac e ia (S ahl and
E ing on, 2017;B amkamp and Lopez, 2015;Lopez and Koch, 2017). These so-called unc ional
memb ane mic odomains (FMMs) a e hough o be o ganised by he bac e ial lo illin p o eins, a e
en iched in isop enoid lipids (Ga cı´a-Fe na
´ndez e al., 2017;Lo
´pez and Kol e , 2010), and can be
ound in so-called De e gen Resis an Memb ane (DRM) ac ions o he memb ane. Since he o -
mula ion o he FMM hypo hesis, FMMs ha e been linked o many p ocesses, such as p o ein sec e-
ion, bio ilm o ma ion, compe ence and cell mo phology (Mielich-Su
¨ss and Lopez, 2015;Mielich-
eLi e diges E e y li ing cell is enclosed by a lexible memb ane made o molecules known as
phospholipids, which p o ec s he cell om ha m ul chemicals and o he h ea s. In bac e ia and
some o he o ganisms, a igid s uc u e known as he cell wall si s jus ou side o he memb ane and
de e mines he cell’s shape.
The e a e se e al p o eins in he memb ane o bac e ia ha allow he cell o g ow by assembling
new pieces o he cell wall. To ensu e hese p o eins expand he cell wall a he igh loca ions,
ano he p o ein known as M eB mo es and o ganizes hem o he app op ia e place in he
memb ane and con ols hei ac i i y. P e ious s udies ha e ound ha ano he class o p o eins
called lo illins a e in ol ed in a anging p o eins and phospholipid molecules wi hin memb anes.
Bac e ia lacking hese p o eins do no g ow p ope ly and a e unable o main ain hei no mal shape.
Howe e , he p ecise ole o he lo illins emained unclea .
He e, Zielin´ ska, Sa ie o e al. used mic oscopy app oaches o s udy lo illins in a bac e ium
known as Bacillus sub ilis. The expe imen s ound ha , in he p esence o lo illins, M eB mo ed
a ound he memb ane mo e quickly (sugges ing i was mo e ac i e) han when no lo illins we e
p esen . Simila esul s we e obse ed when bac e ial cells lacking lo illins we e ea ed wi h a
chemical ha made memb anes mo e ‘ luid’ – ha is, made i easie o he molecules wi hin he
memb ane o a el a ound. Fu he expe imen s ound ha lo illins allowed he phospholipid
molecules wi hin an a i icial memb ane o mo e a ound mo e eely, which inc eases he luidi y o
he memb ane.
These indings sugges ha lo illins make he memb anes o bac e ial cells mo e luid o help
cells expand hei walls and pe o m se e al o he p ocesses. Unde s anding how bac e ia con ol
he componen s o hei memb anes will u he ou unde s anding o how many cu en ly a ailable
an ibio ics wo k and may po en ially lead o he design o new an ibio ics in he u u e.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 2 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
Su
¨ss e al., 2013;Bach and B amkamp, 2013;Dempwol e al., 2012). Cell mo phology de ec s
a e linked o cell wall syn hesis, and analysis o he p o ein con en o Bacillus sub ilis DRMs iden i-
ied se e al PBPs, M eC and o he p o eins in ol ed in cell wall me abolism as well as he wo lo il-
lins, FloA and FloT (Lo
´pez and Kol e , 2010;Bach and B amkamp, 2013;Yepes e al., 2012). FloA
is cons i u i ely exp essed, whe eas FloT is exp essed p ima ily du ing s a iona y g ow h, cell wall
s ess and spo ula ion (Schneide e al., 2015a;Huang e al., 1999;Nicolas e al., 2012). Supe
esolu ion mic oscopy showed ha he lo illins and o he p o eins ound in DRMs do no colocalise
and ha e di e en dynamics (Dempwol e al., 2016), so i is unlikely ha FMMs a e egions in he
memb ane ha o e a a ou able en i onmen in which hese memb ane p o eins a e con inuously
p esen and ac i e. Recen ly, he hypo hesis has been pu o wa d ha FMMs/ lo illins o m a pla -
o m o he o ma ion o unc ional p o ein oligome s, as wo k in S aphylococcus au eus showed
ha mul ime isa ion o Type 7 sec e ion sys ems and PBP2a depends on FMMs (Lopez and Koch,
2017;Ga cı´a-Fe na
´ndez e al., 2017;Mielich-Su
¨ss e al., 2017).
He e, we ha e analysed he ole o lo illins in pep idoglycan syn hesis in B. sub ilis. Ou esul s
show ha , a high g ow h a es, lo illins con ol memb ane luidi y in a manne ha is c i ical o
pep idoglycan syn hesis and M eB dynamics, bu ha e no e ec on PBP oligome isa ion. This esul s
in a new model o lo illin unc ion in he physical o ganisa ion o memb anes du ing as g ow h.
Resul s
Absence o lo illins shi s pep idoglycan syn hesis o di ision Sep a
In p e ious s udies, a double dele ion o loA/ loT was ei he epo ed o su e se e e shape de ec s
and pe u bed memb ane s uc u e (Dempwol e al., 2012), o o no ha e s ong shape de ec s
bu wi h a change in he o e all lipid o de ing o he memb ane (Bach and B amkamp, 2013). We
g ew wild ype and D loAT s ains and analysed exponen ially g owing cells. We did no obse e
s iking shape de ec s bu did see an inc ease in median cell leng h and dis ibu ion o cell leng hs in
he absence o lo illins (Figu e 1A,G). To look a e ec s on pep idoglycan syn hesis, we labelled
cells wi h HADA, a luo escen D-Alanine analogue ha epo s on si es o ac i e pep idoglycan syn-
hesis (Ku u e al., 2012), and wi h luo escen ancomycin (Van-FL), which labels LipidII and pep i-
doglycan con aining pen apep ide side chains (Daniel and E ing on, 2003;Mo ales Angeles
e al., 2017). This e ealed a signi ican accumula ion o pep idoglycan syn hesis s ains a di ision
sep a in he D loAT s ain (Figu e 1A–C). To look a memb ane s uc u e, cells we e labelled wi h
FM4-64, Nile-Red and DiI-C12, which a e lipid dyes ha accumula e in zones en iched in luid lipids
(S ahl e al., 2014). Again, he s ains accumula ed a he sep a in he D loAT s ain, which also
showed some accumula ion o FM4-64 and DiI-C12 in pa ches, sugges ing ha he mo e luid
egions o he memb ane a e coalescing in o la ge egions (Figu e 1A,D–F). The HADA, FM4-64
and Nile-Red measu emen s we e epea ed using a wild ype s ain exp essing endogenous GFP,
allowing simul aneous imaging o bo h s ains on he same slide, and ga e simila esul s, con i ming
ha he obse ed signal inc ease is no due o a ia ion be ween mic ocopy expe imen s (Figu e 1—
igu e supplemen 1A,B). In his mixed-s ain expe imen , Nile-Red labelling a he la e al mem-
b ane was he same be ween wild ype and D loAT s ains, indica ing ha he e is no di e ence in
dye di usion be ween he s ains (Figu e 1— igu e supplemen 1D). Inspec ion o he sep a by
elec on mic oscopy e ealed ha he e was no di e ence be ween he hicknesses o he sep a
be ween he wild ype and D loAT s ain, uling ou ha he inc ease in signal was due o o ma ion
o hicke sep a (Figu e 1— igu e supplemen 1C). The shi o pep idoglycan syn hesis o he di i-
sion si e could hin a s ess in he o e all pep idoglycan syn hesis ou e. This was con i med by
g owing cells a a suble hal concen a ion o os omycin, which limi s syn hesis o LipidII
(Kahan e al., 1974), bu ha does no impac g ow h a e a he concen a ion used. This esul ed
in bulging cells and some lysis, which was exace ba ed in he D loAT s ain (Figu e 1— igu e supple-
men 1F,G). I should be no ed ha he pep idoglycan syn hesis s ess caused by os omycin is no
he same as he s ess caused by he absence o lo illins, as he pheno ypes o wild ype cells wi h
suble hal os omycin a e qui e dis inc om D loAT cells wi hou os omycin.
We uled ou ha he pep idoglycan syn hesis s ess was caused by a change in he olding o
complex o ma ion by PBPs in he absence o lo illins, as he e we e no di e ences in he o e all
PBP-p o iles o Bocillin-FL labelled wild ype o lo illin dele ion s ains (Figu e 1— igu e
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 3 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
Figu e 1. Accumula ion o pep idoglycan syn hesis and memb ane ma e ial a di ision si es in a lo illin mu an . (A) Mo phology o he exponen ially
g owing wild ype (WT) and D loAT s ains labelled wi h HADA, luo escen Vancomycin (Van-FL), FM 4–64, Nile Red, and DiI-C12. Scale ba : 5 mm. (B–F)
Peak in ensi y o HADA (B), Van-FL (C), Nile Red (D), FM4-64 (E) and DiI-C12 (F) labelled di ision si es o he cells shown in (A). Cells om each s ain
(n 100, excep E, n = 60) we e analysed using he Objec J mac o ool PeakFinde ollowed by s a is ical analysis wi h P ism. Signi ican di e ences a e
based on he wo- ailed Mann-Whi ney es (*p<0.05; **p<0.01). (G) Dis ibu ion o he cell leng h o he s ains analysed in (A). S a is ical analysis o he
da a (n = 100, wo ailed Mann-Whi ney es , *p<0.05) was pe o med wi h P ism, esul ing in box plo g aphs.
The online e sion o his a icle includes he ollowing sou ce da a and igu e supplemen (s) o igu e 1:
Sou ce da a 1. Fluo escence in ensi y and cell leng h measu emen s.
Figu e supplemen 1. Con ol expe imen s showing ha di e ences in sep al labeling in ensi y a e no due o mic oscopy se ings, sep um hickness,
o dye di usion.
Figu e supplemen 1—sou ce da a 1. G ow h da a plo ed in FS1F.
Figu e supplemen 1—sou ce da a 2. Da a plo ed in F1F1Sb, F1FS1C, F1FS1.
Figu e 1 con inued on nex page
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 4 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
supplemen 2A). PBP complex o ma ion was analysed using a combina ion o Na i e-PAGE and
SDS-PAGE wi h Bocillin-labelled memb ane ac ions (T ip and Sche e s, 2016) and showed ha
a ious PBPs can be ound in a high-MW complex (no ably PBPs 1, 2, 3 and 4), bu ha complex o -
ma ion is simila in he D loAT s ain (Figu e 1— igu e supplemen 2B). Also, none o he i e unc-
ional GFP-PBPs examined changed hei localisa ion in he D loAT s ain (Figu e 1— igu e
supplemen 2C). O e all, he da a sugges ha in he absence o lo illins, pep idoglycan syn hesis
is a ec ed and ela i ely inc eased a di ision sep a, wi h a concomi an accumula ion o memb ane
dyes ha a e indica i e o highe memb ane luidi y.
The absence o bo h lo illins and PBP1 causes a se e e pheno ype,
linked o a loss o memb ane luidi y
We easoned ha a non-le hal de ec in sep al pep idoglycan syn hesis could e eal mo e abou he
ole o lo illins and cons uc ed a lo illin mu an ha lacks PBP1, a bi unc ional glycosyl ans e ase/
anspep idase ha is equi ed o e icien cell di ision (Sche e s and E ing on, 2004). Simul a-
neous dele ion o pbp1, loA, and loT esul ed in s ong ilamen a ion and delocalisa ion o pep ido-
glycan syn hesis as well as memb ane dyes o pa ches (Figu e 2A,Figu e 2— igu e supplemen
1A). Dele ion o single lo illin genes and PBP1 had simila , albei less se e e e ec s (Figu e 2— ig-
u e supplemen 1B,C). To exclude he possibili y ha an al e a ion o pep idoglycan modi ica ion
esul ed in he delocalisa ion o HADA and Van-FL, we used D-Alanine-D-P opa gylglycine (D-Ala-D-
P a), a clickable dipep ide analogue which is exclusi ely inco po a ed in o pep idoglycan ia LipidII
(Sa ka e al., 2016). D-Ala-D-P a inco po a ion was delocalised in he Dpbp1D loAT s ain, indica -
ing ha pep idoglycan syn hesis i sel is delocalised (Figu e 2— igu e supplemen 1D). So a , ou
expe imen s we e done wi h as g owing cells and Lysogeny B o h (LB) as he g ow h medium.
S ikingly, none o he mu an s ains had an appa en pheno ype when cul i a ed in Spizizen’s mini-
mal medium (SMM, Figu e 2B), and pep idoglycan syn hesis and lipid dyes we e no longe accumu-
la ing a di ision si es in he D loAT s ain (Figu e 2— igu e supplemen 2). SMM has a highe Mg
2+
concen a ion, which is known o escue a ious cell shape mu a ions by inhibi ion o cell wall hyd o-
lysis (Dajko ic e al., 2017). Howe e , he inc ease in Mg
2+
was no su icien o explain he e e sal
o pheno ype as cells g own on LB supplemen ed wi h Mg
2+
(6 mM, concen a ion in SMM, o 20
mM) s ill displayed he elonga ed pheno ype wi h delocalised pep idoglycan syn hesis (Figu e 2—
igu e supplemen 3). This indica ed ha he pheno ypes associa ed wi h he absence o lo illins
a e g ow h- a e and/o nu ien ela ed.
Nex , we de e mined lipid packing o de in he di e en s ains using he luo escen dye Lau -
dan, a epo e o lo illin-media ed lipid o de ing (Bach and B amkamp, 2013). LB-g own cells
lacking lo illins displayed an inc eased gene alised pola isa ion (GP) (Bach and B amkamp, 2013),
indica i e o an o e all inc ease in o de ed lipid packing in he memb ane, bu he e ec o lo illins
on memb ane o de ing comple ely disappea ed when cells we e g own on SMM (Figu e 3). The es-
olu ion ob ained wi h Lau dan does no allow he de ec ion o local di e ences in luidi y be ween
he la e al memb ane and he sep a, bu does epo on o e all lipid o de ing. O e all, lipid o de
was inc eased in cells g own on SMM compa ed o LB (Figu e 3), whe eas he absence o PBP1 had
no signi ican e ec on memb ane luidi y, also no when combined wi h lo illin dele ions (Figu e 3).
The changes in lipid o de ing we e no due o changes in he o e all a y acid composi ion o he
memb anes - he a ios o C17/C15 side chains and iso/an eiso a y acids, which a e indica i e o lu-
idi y (S ahl e al., 2014), we e iden ical o wild ype and D loAT s ains g own on LB, and e y simi-
la o cells g own on SMM (Figu e 3— igu e supplemen 1).
Res o ing memb ane luidi y escues no mal pep idoglycan syn hesis
The GP alues indica ed ha memb anes a e mo e o de ed when cells a e g own on minimal
medium, and his sugges s ha he lo illin-associa ed inc ease in o e all memb ane luidi y is impo -
an o cell shape con ol a high g ow h a es. This was es ed by g owing he s ains lacking lo il-
lins and PBP1 on LB in he p esence o benzyl alcohol, an ex ensi ely used memb ane luidise ha
Figu e 1 con inued
Figu e supplemen 2. Absence o lo illins does no a ec exp ession, oligome isa ion o localisa ion o PBPs.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 5 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
Figu e 2. Cell mo phology and cell wall syn hesis localisa ion is dependen on g ow h condi ions. Mo phology o he WT,D loAT,Dpbp1, and
Dpbp1D loAT s ains g own in (A) ich (LB), (B) minimal (SMM) medium, and in (C) ich medium wi h memb ane luidising condi ions (0.1% benzyl
alcohol, LB+BnOH). Cells we e labelled wi h HADA, and abe an cell shape and pep idoglycan syn hesis a e indica ed wi h a owheads. Panels on he
igh indica e co esponding cell leng h dis ibu ions (n 100). Dis ibu ions we e analysed using Dunn’s mul iple compa ison es s a e K uskal–Wallis.
S a is ically signi ican cell leng h dis ibu ion classes (p<0.001) a e ep esen ed as le e s abo e each g aph – in B and C he e we e no signi ican
di e ences. Scale ba : 4 mm.
The online e sion o his a icle includes he ollowing sou ce da a and igu e supplemen (s) o igu e 2:
Sou ce da a 1. Cell leng h measu emen s.
Figu e supplemen 1. Dele ion o bo h lo illins and PBP1 induces ilamen a ion and delocalisa ion o pep idoglycan syn hesis.
Figu e supplemen 1—sou ce da a 1. Cell leng h measu emen s plo ed in F2F1C.
Figu e supplemen 2. Sep um labelling o wild ype and lo illin mu an cells g own on minimal medium.
Figu e supplemen 2—sou ce da a 1. Fluo escence in ensi y measu emen s plo ed in F2FS2.
Figu e supplemen 3. Filamen a ion and delocalisa ion o pep idoglycan syn hesis in he absence o lo illins and PBP1 is no escued by he addi ion
o magnesium.
Figu e supplemen 4. G ow h cu es and g ow h a es show simila g ow h o w , D loAT,Dpbp1, and Dpbp1D loAT (as well as D loA, D loT,
Dpbp1D loA and Dpbp1D loT) s ains g own on LB o on LB supplemen ed wi h BnOH (0.1% (w/ )).
Figu e supplemen 4—sou ce da a 1. G ow h cu e da a.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 6 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
inc eases memb ane hyd a ion due o diso de ing o memb ane s uc u e (Konopa
´sek e al., 2000).
No ably, he addi ion o benzyl alcohol inc eased memb ane luidi y o simila ex en s in he wild-
ype and he mu an s ains (see Figu e 3C), bu did no a ec he g ow h a es o he s ains (Fig-
u e 2— igu e supplemen 4). The inc ease in memb ane luidi y es o ed no mal cell leng h and
no mal pep idoglycan syn hesis pa e ns o he pbp1/ loA/ loT s ain (Figu e 2C).
In B. sub ilis, he a e o g ow h and o pep idoglycan syn hesis is linked o he speed o M eB
mo emen – in minimal media, he speed o M eB pa ches is educed compa ed o he speed in ich
media (Billaudeau e al., 2017). Analysis o he mo emen o a ully unc ional mRFP uby-M eB
usion (Domı´nguez-Escoba e al., 2011) by ime lapse TIRF (To al In e nal Re lec ion Fluo escence)
mic oscopy, con i med ha M eB pa ch mobili y is highe in cells g own on LB han in cells g own on
SMM, wi h M eB speeds simila o hose epo ed p e iously (Billaudeau e al., 2017;Figu e 4,Fig-
u e 4— ideos 1 and 2). S ikingly, in he absence o lo illins, M eB pa ch mobili y was no ably
dec eased in cells g own on LB, while in SMM g own cells M eB pa ch mobili y was independen o
he p esence o lo illins (Figu e 4,Figu e 4— ideos 3 and 4). Fluidising he memb ane wi h benzyl
alcohol, which does no al e he g ow h a e, almos comple ely es o ed M eB mobili y in LB g own
cells (Figu e 4,Figu e 4— ideos 5 and 6). These esul s indica e ha he M eB pa ch mobili y is
no only con olled by g ow h a e, bu also by memb ane luidi y. Thus, in as g owing cells wi h
dec eased memb ane luidi y he e is a dec ease in elongasome media ed pep idoglycan syn hesis,
Figu e 3. Flo illins inc ease o e all memb ane luidi y a high g ow h a e. Changes in o e all memb ane luidi y we e assessed by Lau dan mic oscopy
in cells g own on LB (A), SMM (B) and LB+BnOH (C). Mic og aphs show colou -coded gene alised pola isa ion (GP) maps in which ed indica es egions
o dec eased luidi y (scale ba : 4 mm). Co esponden heo e ical GP measu emen s in he g aphs a y om 1 (mo e luid) o 1 (less luid). Signi ican
s a is ical di e ences acco ding o Dunn’s mul iple compa ison es s a e K uskal–Wallis a e ep esen ed as le e s abo e each g aph in panel (A). Da a
labelled ‘A’ a e signi ican ly di e en om da a labelled ‘B’; da a wi h he same le e a e no signi ican ly di e en . No s a is ically signi ican di e ence
was obse ed o he da a in panels (B) and (C) (p<0.001; n 150, wo biological eplica es).
The online e sion o his a icle includes he ollowing sou ce da a and igu e supplemen (s) o igu e 3:
Sou ce da a 1. GP measu emen .
Figu e supplemen 1. Fa y acid composi ion analysis.
Figu e supplemen 1—sou ce da a 1. Fa y acid composi ion da a.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 7 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
e lec ed by he educ ion o M eB mobili y. This i s wi h an obse ed inc ease in pep idoglycan syn-
hesis a he di ision si e which may ac as a compensa o y mechanism.
Flo illin inc eases luidi y o model memb anes in i o
To assess whe he he in luence o lo illins on memb ane luidi y is di ec , we de e mined he mem-
b ane luidi y o model memb anes wi h pu i ied lo illin using solid-s a e NMR (ssNMR).
2
H ssNMR
is a biophysical ool ha assesses lipid mobili y in na i e-like model memb anes on he a omic le el,
by moni o ing he ca bon-deu e ium o de pa ame e o a deu e a ed lipid along he acyl chain
(he e POPC-d31) (Molugu e al., 2017;Leg and e al., 2019). We pu i ied B. sub ilis FloT and
es ed he impac o FloT on he memb ane, when econs i u ed in POPC-d31 liposomes (Schema i-
cally depic ed in Figu e 5A). FloT dec eases he spec al wid h o he
2
H quad upola spli ing,
e lec ing an inc ease in mo ion on he a omic scale (Figu e 5B). The
2
H spec um encodes he local
o de pa ame e S
CD
o he ca bon-deu e ium in absence and in p esence o FloT. S ikingly, FloT
Figu e 4. M eB speed is linked o memb ane luidi y. (A) The M eB speed in di e en s ain backg ounds and g ow h condi ions was analysed by ime-
lapse TIRF mic oscopy. Sca e plo o he speed o pa ches ob ained om indi idual acks in 5 di e en cells a e ep esen ed pe usion and
condi ion. A e age speeds a e shown; e o ba s indica e he s anda d de ia ion. Signi ican s a is ical di e ences acco ding o Dunn’s mul iple
compa ison es s a e K uskal–Wallis a e ep esen ed (p<0.001). (B) Rep esen a i e kymog aphs showing as and slow mo ing pa ches o mRFP uby-
M eB in B. sub ilis cells lacking endogenous m eB (WT) o m eB and loAT (D loAT). See Figu e 4— ideos 1–6 o co esponding aw image se ies.
The online e sion o his a icle includes he ollowing ideo and sou ce da a o igu e 4:
Sou ce da a 1. M eB pa ch mobili y measu emen s de e mined by TIRFM.
Figu e 4— ideo 1. Visualisa ion o xylose inducible m pRuby-M eB pa ches dynamics (s ain 4070) du ing exponen ial g ow h in LB medium a 37˚C by
TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo1
Figu e 4— ideo 2. Visualisa ion o xylose inducible m pRuby-M eB pa ches dynamics (s ain 4070) du ing exponen ial g ow h in SMM medium a 37˚C
by TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo2
Figu e 4— ideo 3. Visualisa ion o xylose inducible m pRuby-M eB in D loAT pa ches dynamics (s ain 4076) du ing exponen ial g ow h in LB medium
a 37˚C by TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo3
Figu e 4— ideo 4. Visualisa ion o xylose inducible m pRuby-M eB in D loAT pa ches dynamics (s ain 4076) du ing exponen ial g ow h in SMM
medium a 37˚C by TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo4
Figu e 4— ideo 5. Visualisa ion o xylose inducible m pRuby-M eB pa ches dynamics (s ain 4070) du ing exponen ial g ow h in LB medium supple-
men ed wi h BnOH (0.1%) a 37˚C by TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo5
Figu e 4— ideo 6. Visualisa ion o xylose inducible m pRuby-M eB in D loAT pa ches dynamics (s ain 4076) du ing exponen ial g ow h in LB medium
supplemen ed wi h BnOH (0.1%) a 37˚C by TIRF mic oscopy.
h ps://eli esciences.o g/a icles/57179# ig4 ideo6
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 8 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
has an impo an impac on he o de pa ame e along he en i e acyl chain. I is ema kable ha he
p o ein signi ican ly dec eases he o de pa ame e S
CD
, eaching e en he inne ca bon a oms o
he acyl chain, indica ing a di e en packing beha iou and inc eased memb ane luidi y upon in e -
ac ion wi h FloT (Figu e 5B). The s ong luidising e ec desc ibed o FloT is no ably di e en om
he e ec s o he p o eins ha e when econs i u ed in o liposomes, such as plan emo ins
(Leg and e al., 2019) o he memb ane binding pep ide o he non ecep o y osine kinase S c
(Scheid and Hus e , 2009). The aniso opic lineshape o he
31
P spec a indica es ha he mem-
b ane is in he lamella phase as expec ed o POPC a he chosen empe a u e (298K) (Hus-
e , 2014). Upon in e ac ions wi h FloT he lamella phase emains in ac wi h o ma ion o a ew
smalle objec s, indica ing ha he o e all liposome s uc u e is no a ec ed and ha i s phase is
main ained (Figu e 5— igu e supplemen 1).
Discussion
Ou da a p o ide e idence ha lo illins play a di ec ole in con olling memb ane luidi y and ha
memb ane luidi y is c i ical o pep idoglycan syn hesis a ce ain g ow h condi ions. In i o, lo il-
lins enhance he luidi y o a model memb ane, and in i o, he memb anes o as g owing lo illin-
mu an cells a e less luid e en hough he a y acid composi ion in hese cells is iden ical. The e-
o e, we p opose ha he e ec o lo illins on memb ane luidi y is di ec , h ough a change in he
packing beha iou o he lipids esul ing in an e icien sepa a ion o s a es o liquid o de ed and dis-
o de ed lipid domains in he memb ane bilaye (Bach and B amkamp, 2013). We ound ha
Figu e 5. Lipid o de ing o FloT p obed by
2
H solid-s a e NMR. (A) Wide-line
2
H spec a o POPC-d31 liposomes wi h o wi hou FloT a a lipid- o-
p o ein mola a io o 25:1 acqui ed a 298 K. (B) E ec o FloT on he C-
2
H o de pa ame e s o he PC acyl chain. De-Pake-ing and simula ions we e
applied on he
2
H solid-s a e NMR spec a o de e mine accu a ely indi idual quad upola spli ings. O de pa ame e s o POPC-d31 acyl chain we e
de i ed om expe imen al quad upola spli ings and plo ed as a unc ion o he labelled ca bon posi ion. Inse : schema ic depic ion o a liposome
wi h added FloT which a aches o he memb ane ia a hai pin loop (Bach and B amkamp, 2015).
The online e sion o his a icle includes he ollowing igu e supplemen (s) o igu e 5:
Figu e supplemen 1.
31
P solid-s a e NMR expe imen s o POPC liposomes wi h o wi hou FloT a a lipid- o-p o ein mola a io o 25:1.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 9 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
Bocillin labelling
Cells we e g own un il an OD600 o 0.4–0.5, and washed wice wi h PBS. Nex , samples we e esus-
pended in 50 ml PBS con aining Bocillin-FL (5 mg/ml) and incuba ed a oom empe a u e o 10 min.
Subsequen ly cells we e ha es ed, lysed by sonica ion and cell- ee ex ac s we e p epa ed. Sam-
ples, equalised o cul u e OD, we e p epa ed wi h SDS-PAGE sample bu e and un on a 12% SDS-
PAGE gel. Fluo escen bands we e isualised using a Typhoon T io (GE Heal hca e) scanne .
Isola ion o memb anes
Memb ane isola ion was adap ed om Schneide e al., 2015b. B ie ly, cells we e g own un il an
OD600 o 0.4–0.5, cell ac ions we e collec ed and esuspended in PBS wi h Lysozyme (1 mg/ml),
EDTA (5 mM), 1/10 able cOmple e p o ease inhibi o (Roche), and DNAse (5 mg/ml) and incuba ed
o 30 min on ice. Samples we e sonica ed, cell which did no lyze we e spun down (8000 pm, 2
min, 4˚C), and he supe na an ac ion was cen i uged a 4˚C and 40000 pm o 1 h . The mem-
b ane pelle was dissol ed in ACA750 bu e (750 mM aminocap oic acid, 50 mM Bis-T is, pH 7.0) o
a inal p o ein concen a ion o 1 mg/ml. Memb anes we e solubilised o e nigh a 4˚C in 1% (w/ )
dodecylmal oside (DDM) and ei he used di ec ly o s o ed a 20˚C.
Blue na i e PAGE (BN-PAGE)
The expe imen was pe o med as desc ibed (T ip and Sche e s, 2016). Samples we e p epa ed by
mixing sample bu e (0.1% Ponceau S, 42.5% Glyce ol) wi h solubilised memb anes in a 1:3 a io.
Samples we e esol ed on a mini-PROTEAN TGX S ain-F ee g adien gel (4–15%, BioRad) using
ca hode (50 mM T icine and 15 mM BisT is), and anode (50 mM BisT is pH 7.0) bu e s. The No ex
Na i eMa k Uns ained P o ein S anda d ma ke was used as a Mw ma ke .
Second dimension SDS PAGE (2D SDS-PAGE)
A lane o in e es was excised om he Na i e-PAGE gel and immobilised ho izon ally on op o a
SDS-PAGE gel (5% s acking, 12% esol ing). The excised agmen was lanked wi h a piece o Wha -
man pape soaked wi h PageRule P es ained P o ein Ladde . The gel agmen o be esol ed in
he second dimension was opped wi h a mix o 1% (w/ ) LowTempe a u e aga ose, 0.5% (w/ ) SDS
and b omophenol blue. A e he aga ose had solidi ied, s anda d SDS-PAGE elec opho esis was
pe o med.
TEM
Cul u es we e ha es ed by cen i uga ion and a small amoun o pelle was placed on a coppe
dish. A 400 coppe mesh g id and a 75 mm ape u e g id was placed on op o he cells o c ea e a
hin laye . The sandwiched cells we e plunged apidly in o liquid p opane. Sandwiches we e hen dis-
assembled and placed on ozen eeze-subs i u ion medium con aining 1% osmium e oxide, 0.5%
u anyl ace a e and 5% wa e in ace one. Cells we e dehyd a ed and ixed using he apid eeze sub-
s i u ion me hod (McDonald, 2014). Samples we e embedded in epon and ul a hin sec ions we e
collec ed on o m a coa ed and ca bon e apo a ed coppe g ids and inspec ed using a CM12 (Phi-
lips) ansmission elec on mic oscope. Fo each s ain 70 andom sep a we e imaged wi h pixel es-
olu ion o 1.2 nm. Using ImageJ he cell wall hickness o each sep um was measu ed a 4 places
om which he a e age was aken.
S a is ical analysis
Each se o mic og aphs o be analysed was imaged wi h he same exposu e ime. Fo he sep um
in ensi y analysis o HADA, FM4-64 and Nile Red, he wild ype s ain (exp essing GFP) and he
D loAT s ains we e mixed, labelled and imaged on he same aga ose pad. In ensi y o he luo es-
cen ly labelled sep a was measu ed using he Objec J mac o ool PeakFinde (h ps://sils. nwi.u a.
nl/bcb/objec j/examples/PeakFinde /peak inde .h ml) (Vische e al., 2015). A pe pendicula line
was d awn ac oss he sep al plane, he backg ound in ensi y was emo ed esul ing in a maximum
peak in ensi y. The numbe o sep a compa ed was indica ed o e e y indi idual expe imen . Popu-
la ions we e compa ed using he non-pa ame ic Mann-Whi ney es . The null hypo hesis was es ed
wi h he p alue o 0.05. The s a is ical analyses and hei g aphical ep esen a ion (box plo s) we e
gene a ed wi h G aphPad P ism 8.1 (San Diego, Cali o nia, USA). Box plo s show he median and
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 16 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
he in e qua ile ange (box), he 5 h and 95 h pe cen ile (whiske s). Lau dan luo escence gene al-
ised pola isa ion, cell leng h and M eB speed s a is ical analyses we e pe o med using K uskal-
Wallis wi h Dunn’s mul iple compa ison pos -hoc es .
Fa y acid composi ion analysis
The a y acid composi ion o B. sub ilis wild- ype cells and he lo illin/PBP mu an s was analysed
wi h gas ch oma og aphy as a y acid me hyl es e s. Cells o he analyses we e g own a 37˚C in LB
o SMM un il mid-exponen ial (OD
600
~0.5), ha es ed (6000 pm, 10 min, 4˚C) and washed wi h 100
mM NaCl. Nex , he cells we e eeze d ied a 50˚C, 0.012 mba o a minimum o 18 h . All analy-
ses we e ca ied ou on biological duplica es by he Iden i ica ion Se ice o he DSMZ, B aunsch-
weig, Ge many.
Sample p epa a ion o solid-s a e NMR
FloT was essen ialy pu i ied as desc ibed (Bach and B amkamp, 2013), in solubilised o m, and
s o ed in bu e A (50 mM T is HCl pH 7.5, 150 mM NaCl, 5 mM MgCl
2
) supplemen ed wi h 0.05%
T i on X-100.
Liposomes con aining POPC-d31 we e p epa ed by mixing app op ia e lipid powde s in o ganic
sol en s (chlo o o m/me hanol, 2:1 a io). Sol en s we e e apo a ed unde a low o N
2
o ob ain a
hin lipid ilm. Lipids we e ehyd a ed wi h ul apu e wa e be o e lyophilisa ion o e nigh . The lipid
powde was hyd a ed wi h an app op ia e amoun o bu e A wi h 10% glyce ol and homogenised
by h ee cycles o o exing, eezing (liquid ni ogen, 196˚C, 1 min) and hawing (40˚C in a wa e
ba h, 10 min). This p o ocol gene a ed a milky suspension o mic ome e -sized mul ilamella esicles.
FloT was solubilised in Bu e A supplemen ed wi h 0.05% T i on X-100 and added o p e o med lip-
osomes and incuba ed o 1 h a oom empe a u e. A dialysis s ep was hen pe o med agains
Bu e A a 4˚C unde agi a ion o emo e he de e gen . Samples we e cen i uged a 100,000 g a
4˚C o 1 h o pelle he p o eoliposomes.
2
H solid-s a e NMR spec a we e eco ded o liposomes
in he p esence o absence o FloT a a lipid/p o ein a io o 25:1 a 298 K.
Solid-s a e NMR
2
H NMR spec oscopy expe imen s we e pe o med using a B uke A ance III 500 MHz WB (11.75 T)
spec ome e . They we e eco ded on
2
H-labelled POPC a 76.77 MHz wi h a phase-cycled quad u-
pola echo pulse sequence (90˚x- -90˚y- -acq). Acquisi ion pa ame e s we e as ollows: spec al win-
dow o 500 kHz o 2H NMR spec oscopy, p/2 pulse wid h o 3.90 ms o
2
H, in e pulse delays ( )
we e o 40 ms, ecycled delays o 1.3 s o 2H; 3000 and 8000 scans we e used o
2
H NMR spec os-
copy on liposomes and liposomes wi h FloT, espec i ely. Spec a we e p ocessed using a Lo en zian
line b oadening o 300 Hz o
2
H NMR spec a be o e Fou ie ans o ma ion om he op o he
echo. Samples we e equilib a ed o 30 min a a gi en empe a u e be o e da a acquisi ion. All spec-
a we e p ocessed and analysed using B uke Topspin 3.2 so wa e. Spec al momen s we e calcu-
la ed o each empe a u e using he NMR Depake 1.0 c1 so wa e [Copy igh (C) 2009 Se´ bas ien
Buchoux]. O ien a ional o de pa ame e s (SCD) we e calcula ed om expe imen al quad upola
spli ings (DnQ) as desc ibed in Hus e , 2014. Fo
31
P ssNMR, we applied a s a ic Hahn spin echo
sequence a he
31
P equency o 162 MHz on a 400 MHz (9.4T) B uke A ance III HD spec ome e ,
wi h a 90pulse o 8 ms, a delay o 40 ms, a ecycle delay o 5 s, a spec al window o 400 ppm and a
numbe o scans o 4000 and 3400 was used on liposomes and liposomes wi h FloT, espec i ely.
Spec a we e p ocessed using a Lo en zian line b oadening o 100 Hz.
Acknowledgemen s
We hank Hen ik S ahl o discussions and sha ing o unpublished da a, Ru Ca ballido-Lopez o
s ain RWBS5 and Luiza Mo awska and Osca Kuipe s o he P nB-g p plasmid.
This wo k was unded by NWO g an 864.09.010 (DJS), DFG g an s BR 2915/4–1; INST 86/1452–
1 (MB), ERC s a ing g an 757913; NWO g an 721.014.008 (AKHH); PhD ellowships DAAD-GSSP
o AS; and SFRH/BD/78061/2011- POPH/FSE/FCT o ASB.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 17 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
Addi ional in o ma ion
Funding
Funde G an e e ence numbe Au ho
Nede landse O ganisa ie oo
We enschappelijk Onde zoek
Vidi 864.09.010 Di k-Jan Sche e s
Deu sche Fo schungsge-
meinscha
BR 2915/7-1 Ma c B amkamp
Deu sche Fo schungsge-
meinscha
INST 86/1452-1 Ma c B amkamp
Nede landse O ganisa ie oo
We enschappelijk Onde zoek
721.014.008 Anna KH Hi sch
Eu opean Resea ch Council s a ing g an 757913 Anna KH Hi sch
Deu sche Akademische Aus-
auschdiens
PhD ellowship DAAD-GSSP Abigail Sa ie o
Fundac¸a
˜o pa a a Cie
ˆncia e a
Tecnologia
PhD ellowship SFRH/BD/
78061/2011- POPH/FSE/
FCT
Anabela de Sousa Bo ges
The unde s had no ole in s udy design, da a collec ion and in e p e a ion, o he
decision o submi he wo k o publica ion.
Au ho con ibu ions
Aleksand a Zielin´ ska, Concep ualiza ion, Da a cu a ion, Fo mal analysis, Supe ision, In es iga ion,
Visualiza ion, Me hodology, W i ing - o iginal d a , W i ing - e iew and edi ing; Abigail Sa ie o,
Concep ualiza ion, Da a cu a ion, Fo mal analysis, In es iga ion, Visualiza ion, Me hodology, W i ing
- o iginal d a , W i ing - e iew and edi ing; Anabela de Sousa Bo ges, Concep ualiza ion, Funding
acquisi ion, In es iga ion, Visualiza ion, Me hodology; Denis Ma inez, Melanie Be bon, Fo mal anal-
ysis, In es iga ion, Me hodology; Joe
¨l R Roelo sen, In es iga ion; Alwin M Ha man, Anna KH Hi sch,
Resou ces; Rinse de Boe , Fo mal analysis, In es iga ion; Ida J Van de Klei, Supe ision, In es iga-
ion; Bi gi Habens ein, Concep ualiza ion, Supe ision, In es iga ion, Me hodology, W i ing - o igi-
nal d a ; Ma c B amkamp, Di k-Jan Sche e s, Concep ualiza ion, Fo mal analysis, Supe ision,
Funding acquisi ion, W i ing - o iginal d a , P ojec adminis a ion, W i ing - e iew and edi ing
Au ho ORCIDs
Ida J Van de Klei h ps://o cid.o g/0000-0001-7165-9679
Ma c B amkamp h p://o cid.o g/0000-0002-7704-3266
Di k-Jan Sche e s h ps://o cid.o g/0000-0002-9439-9168
Decision le e and Au ho esponse
Decision le e h ps://doi.o g/10.7554/eLi e.57179.sa1
Au ho esponse h ps://doi.o g/10.7554/eLi e.57179.sa2
Addi ional iles
Supplemen a y iles
.T anspa en epo ing o m
Da a a ailabili y
All da a gene a ed o analysed du ing his s udy a e included in he manusc ip and suppo ing iles.
Zielin´ska e al. eLi e 2020;9:e57179. DOI: h ps://doi.o g/10.7554/eLi e.57179 18 o 21
Resea ch a icle Cell Biology Mic obiology and In ec ious Disease
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