Recei ed: 23 Ma ch 2024. Re ised: 23 Ap il 2024. Accep ed: 29 Ap il 2024.
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pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
The ISME Jou nal, 2024, 18(1), w ae077
h ps://doi.o g/10.1093/ismejo/w ae077
Ad ance access publica ion: 2 May 2024
O iginal A icle
Side opho es and compe i ion o i on go e n
myxobac e ial p eda ion dynamics
F ancisco Ja ie Con e as-Mo eno1,Au elio Mo aleda-Muñoz1,F ancisco Ja ie Ma cos-To es1,Vi ginia Cuélla 2,Ma ía José So o2,
Juana Pé ez1, José Muñoz-Do ado1,*
1Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada, E-18071 G anada, Spain
2Depa amen o de Bio ecnología y P o ección Ambien al, Es ación Expe imen al del Zaidín, CSIC, E-18008 G anada, Spain
*Co esponding au ho : José Muñoz-Do ado, Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada. A da. Fuen enue a s/n, E-18071
G anada, Spain. Email: jdo ado@ug .es
Abs ac
Bac e ial p eda o s a e decisi e o ganisms ha shape mic obial ecosys ems. In his s udy, we in es iga ed he ole o i on and
side opho es du ing he p eda o y in e ac ion be ween wo hizosphe e bac e ia: Myxococcus xan hus, an epibio ic p eda o , and
Sino hizobium melilo i, a bac e ium ha es ablishes ni ogen- ixing symbiosis wi h legumes.The esul s show ha i on enhances he
mo ili y o he p eda o and acili a es i s p eda o y capabili y, and ha in oxica ion by i on is no used by he p eda o o p ey, al hough
oxida i e s ess inc eases in bo h bac e ia du ing p eda ion. Howe e , compe i ion o i on plays an impo an ole in he ou come
o p eda o y in e ac ions. Using combina ions o p eda o and p ey mu an s (nonp oduce s and o e p oduce s o side opho es), we
ha e in es iga ed he impo ance o compe i ion o i on in p eda ion. The esul s demons a e ha he compe i o ha , ia he
p oduc ion o side opho es, ob ains su icien i on o g ow h and deple es me al a ailabili y o he opponen will p e ail in he
in e ac ion. Consequen ly, i on luc ua ions in soils may modi y he composi ion o mic obial communi ies by al e ing he ac i i y o
myxobac e ial p eda o s. In addi ion, side opho e o e p oduc ion du ing p eda ion can al e soil p ope ies, a ec ing he p oduc i i y
and sus ainabili y o ag icul u al ope a ions.
G aphical abs ac
Keywo ds: Myxococcus xan hus, Sino hizobium melilo i, i on compe i ion, myxochelins, hizobac in 1021
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2|Con e as-Mo eno e al.
In oduc ion
Mic op eda o s shape and main ain he di e si y o mic obial
communi ies, enhance plan heal h and yield, and in luence
ood webs [1–9]. In aqua ic and soil ecosys ems, myxobac e ia
a e majo mic op eda o s ha play impo an ecological oles
because hey o m a nexus be ween he in e ac ions o all
mic oo ganisms in hese habi a s [10–12]. To kill and lyse o he
mic oo ganisms, myxobac e ia use an epibio ic g oup a ack
s a egy [13]. In his s udy, we used he model myxobac e ium
Myxococcus xan hus as a p eda o , which employs a combina ion
o weapons o p ey, wi h some unc ioning a a dis ance and
o he s equi ing close con ac ; consequen ly, mo ili y is also
impo an du ing p eda ion [14–22].As p ey,we used Sino hizobium
melilo i, a ni ogen- ixing bac e ium ha es ablishes symbio ic
ela ionships wi h legumes and plays an impo an ole in
ag icul u al sys ems by enhancing soil e ili y [23].
Mos o ganisms need o main ain homeos asis o se e al
me al ions because hey a e equi ed o i al cellula unc ions
bu a e oxic a high le els [24–26]. The e o e, p eda o s migh
exploi his dual ole o ake ad an age o he in e ac ion, ei he
by deple ing essen ial me als o he p ey o by in oxica ing
hem wi h an excess. In p o ozoan p eda ion, me als ha e
been implica ed in killing p ey [27]. Fo ins ance, Dic yos elium
discoideum uses Cu(I) o inc ease he amoun o eac i e oxygen
species (ROS) inside he phagosome o kill bac e ia [28]. Howe e ,
s udies on he implica ions o essen ial me als in bac e ial
p eda ion a e sca ce. Coppe has been s udied in he p eda o s
Cup ia idus neca o and M. xan hus [28,29], and i could be
hypo hesized ha p eda o y bac e ia also use i on o p ey.
The main mechanism used by bac e ia o i on up ake is
he p oduc ion o side opho es, which a e small and di e se
molecules wi h a high a ini y o e ic ions [31]. In G am-
nega i e bac e ia, he up ake o i on–side opho e complexes
( e iside opho es) is ca ied ou by a anspo e p o ein loca ed
in he ou e memb ane, e med TonB-dependen anspo e
(TBDT), because i is ene gized by he TonB sys em (Fig. S1)[31,
32]. Gene ally, he exp ession o genes in ol ed in side opho e
biosyn hesis and e iside opho e anspo is egula ed by a
ep esso o he Fu (Fe ic Up ake Regula o ) amily, al hough in
some bac e ia,such as hizobia, he i on- esponsi e egula o s a e
I and Ri A [32]. To bind o he ope a o , Fu and Ri A ep esso s
equi e Fe(II), so ha , in he absence o his me al, hey a e
inac i a ed and genes unde hei con ol a e exp essed [31–35].
Al hough he e is much in o ma ion abou side opho e p oduc-
ion and up ake in se e al bac e ia, he ecological ole o hese
seconda y me aboli es emains unexplo ed. To da e, s udies on
i on and p eda ion ha e e ealed ha M. xan hus up egula ed he
exp ession o side opho es and deple es he i on supply o S ep-
omyces coelicolo [36]. These i on- es ic ed condi ions a e espon-
sible o he o e p oduc ion o he an ibio ic ac ino hodin in p ey
[36,37]. O he analyses ha e e ealed ha se e al myxobac e-
ial p eda o s (M. xan hus, Cys obac e e ugineus) and p ey (S.
melilo i,Mic ococcus lu eus,Esche ichia coli, Pseudomonas pu ida,and
Pseudomonas ae uginosa) also up egula e he exp ession o genes
in ol ed in side opho e biosyn hesis when hey in e ac [38–42].
Howe e , he ole o i on and side opho es in he dynamics o
bac e ial p eda ion has no been ho oughly add essed.
To in es iga e he impac o i on and side opho es on he
p eda ion o M. xan hus on S. melilo i,combina ions o p eda o and
p ey mu an s wi h al e ed side opho e p oduc ion (ei he nonp o-
duce s o o e p oduce s) and up ake we e assayed. The esul s
ha e e ealed ha he s ain wi h he abili y o ge enough supply
o i on and deple e he me al o he compe i o will p edomina e.
The e o e, he a ailabili y o i on will be decisi e in he ou come
o he in e ac ion. As a esul , side opho e o e p oduc ion du ing
p eda o y in e ac ions will ha e an impac on he soil mic obiome
and i s p ope ies.
Ma e ials and me hods
Bac e ial s ains, plasmids, and g ow h
condi ions
Bac e ial s ains and plasmids used in his s udy a e lis ed in
Table S1.M. xan hus s ains we e g own in CTT medium [43]a
30◦C. When needed, 100 μg/ml X-gal (5-b omo-4-chlo o-3-indolyl-
β-D-galac opy anoside) and/o 220 μMFeCl
3, o p e en he p o-
duc ion o hizobac in 1021 (Rz1021) [44], we e added. S. melilo i
s ains we e g own a 30◦CinCTT,TY[45], o MM media [46]. Low-
i on MM con ained 2.2 μMFeCl
3, and i was used o he selec ion
o he i A mu an . E. coli s ains we e g own in LB medium [47]
a 37◦C. An ibio ics we e added, as app op ia e, a he ollowing
inal concen a ions: o M. xan hus, kanamycin 80 μg/ml; o S.
melilo i, s ep omycin 200 μg/ml, kanamycin 200 μg/ml, neomycin
100 μg/ml, and e acycline 10 μg/ml; and o E. coli, s ep omycin
50 μg/ml, kanamycin 50 μg/ml, and e acycline 10 μg/ml.
Cons uc ion o he M. xan hus and S. melilo i
mu an s
De ails o he s ains used in his s udy a e shown in Table S1.
M. xan hus in- ame dele ion mu an s we e gene a ed using he
pBJ113 ec o [48] and he p ime s lis ed in Table S2. Plasmids
(Table S1) we e in oduced in o he M. xan hus wild ype (Mx_WT)
by elec opo a ion o gene a e single mu an s. To ob ain he dou-
ble mu an mxcG_ u A, plasmid pFJCMmxcG (Table S1) was elec-
opo a ed in o he u A mu an . S ains we e selec ed as p e i-
ously desc ibed [49].
The in- ame dele ion o he S. melilo i i A gene was gene a ed
by o e lap ex ension polyme ase chain eac ion (PCR) [50] using
p ime s SmRi A1 o SmRi A4 (Table S2) and he suicide plasmid
pK18 mobsacB, yielding plasmid pK18- i A (Table S1). This con-
s uc ion was in oduced in o he wild- ype S. melilo i Rm1021
(Sm_WT) s ain ia conjuga ion by bipa en al ma ing using he E.
coli mobilizing s ain S17-1, and allele eplacemen e en s we e
selec ed as desc ibed p e iously [51]. To a oid he oxic e ec s
caused by i on in pu a i e i A mu an s, he las c osso e e en
was selec ed in low-i on MM. The h A mu an was ob ained
by ans e ing he h A::Tn5 mu a ion om s ain 2011 h A1 o
Sm_WT by phage M12 ansduc ion [52]. Simila ly, he hbA_ i A
mu an was ob ained by ans e ing he hbA::Tn5lac mu a ion
om he hbA mu an o he i A mu an .
Cons uc ion o M. xan hus and S. melilo i s ains
ha bo ing lacZ usions, and β-galac osidase
assays
A plasmid ha bo ing a usion be ween he M. xan hus mxcG gene
and lacZ was cons uc ed using ec o pKY481 [53]and he
oligonucleo ides lis ed in Table S2 as p ime s. The BamHI si e
in he p ime was in oduced a he s a codon o he M. xan hus
gene and in ame wi h he BamHI si e exis ing in he lacZ gene
o plasmid pKY481. This plasmid was in oduced in o M. xan hus
by elec opo a ion, and he s ains we e selec ed as p e iously
desc ibed [49].
To ob ain a ansc ip ional usion o he S. melilo i p omo e
o he h X hbABCDEF ope on o lacZ, a DNA agmen ups eam
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I on in bac e ial p eda ion |3
o h X was PCR ampli ied using Sm_WT genomic DNA and he
p ime s lis ed in Table S2. The esul ing amplicon was cloned in o
pMP220 o gi e he plasmid pMP hBIO,which was used o gene a e
s ains ha bo ing he hb-lacZ usion (Table S1).
Fo β-galac osidase ac i i y analyses, M. xan hus and S. melilo i
we e ea ed as shown in Fig. S2A. Blue colo de elopmen , esul -
ing om β-galac osidase ac i i y in ep esen a i e samples, was
eco ded using an Olympus (Tokyo, Japan) SZX7 dissec ing mic o-
scope equipped wi h a DP72 digi al came a and analyzed using
he Olympus Cell∧F so wa e. β-Galac osidase speci ic ac i i y o
p eda o and p ey in pu e cul u es was quan i ied as p e iously
epo ed [49].
P eda ion expe imen s
Two di e en ypes o assays we e used o s udy p eda ion, which
ha e been e med as dis ance p eda ion assays (DPAs) [16]and
o e lapping p eda ion assays (OPAs) (Fig. S2). OPAs ha e been
ca ied ou as p e iously epo ed wi h modi ica ions [54]. DPAs
we e used o quali a i e gene exp ession, mo ili y, and p eda ion
analyses. When he quan i ica ion o p eda ion was equi ed,
OPAs we e chosen. Due o he low p eda o cell numbe used in
OPAs compa ed wi h ha o he p ey (Fig. S2B), when p eda o
doubling imes we e signi ican ly longe , a DPA app oach was
used o quali a i ely analyze he p eda o y e iciency. No e ha
OPAs do no allow he obse a ion o he ole o mo ili y because
p eda ion ini ia es immedia ely a e spo ing, whe eas in DPAs,
cells equi e ∼24 h o con ac .
Quan i ica ion o M. xan hus and S. melilo i by
d ople digi al PCR
Genomic DNA was ex ac ed om he cells ( h ee d ops pe epli-
ca e), quan i ied using a NanoD op ND-2000 spec opho ome-
e (NanoD op Technologies, Wilming on, DE, USA), and diges ed
wi h SmaI. D ople digi al PCR (ddPCR) was hen conduc ed using
he p ime s and p obes lis ed in Table S2. The ddPCR eac ion
con ained 12 μl o ddPCR Supe mix o p obes (no dUTP) (Bio-
Rad, He cules, CA, USA), 450 mM each o M. xan hus p ime s,
230 nM each o S. melilo i p ime s, 250 nM o M. xan hus p obe,
150 nM o S. melilo i p obe, 5 ng o genomic DNA om each sample
es ed, and molecula -g ade wa e o a o al olume o 22 μl. The
PCR eac ion mix u e was loaded in o DG32 Au oma ed D ople
Gene a o Ca idges (Bio-Rad),and d ople s we e o med wi h he
Au oma ed D ople Gene a o (Bio-Rad). PCR was pe o med in a
T100 he mal cycle (Bio-Rad) unde he ollowing condi ions: one
dena u a ion ho -s a cycle a 95◦C o 10 min,40 cycles o dena -
u a ion a 96◦C o 30 s and annealing a 61◦C o 2 min,and a inal
ex ension s ep a 98◦C o 10 min. All he s eps we e pe o med a
a amp a e o 2◦C/s. Analysis o QX200 D ople Reade (Bio-Rad)
da a was pe o med using QX Manage S anda d Edi ion so wa e
(Bio-Rad) o ack and analyze he luo escen d op dis ibu ion
and posi i e de ec ion h eshold eadings. P ime s and p obes o
each o ganism we e designed a he end po ion o he eplica ion
o k o ensu e ha gene and ch omosome copy numbe s we e
simila (Table S2).
Assay o M. xan hus mo ili y
M. xan hus s ains we e g own and spo ed as shown in Fig. S2A.
The diame e o h ee colonies was measu ed e e y 24 h o
1 week.
Gene a ion ime de e mina ion
Bac e ial s ains we e g own in CTT b o h o an op ical densi y a
600 nm (OD600) o 1. Nex , p eda o and p ey cul u es we e dilu ed
in CTT b o h o an OD600 o 0.05. Flasks om h ee eplica es o
each condi ion we e incuba ed wi h shaking a 30◦C. Cell g ow h
was measu ed spec opho ome ically a OD600 e e y 2 h, and he
gene a ion ime was de e mined du ing he exponen ial g ow h
phase.
Blue ch ome azu ol S (CAS) aga assay o
side opho e de ec ion
Bac e ia we e g own and concen a ed o an OD600 o 15. D ops o
10 μl o he bac e ial suspensions we e deposi ed on o he su ace
o CTT blue ch ome azu ol S (CAS) aga pla es [55]. Pla es om
h ee eplica es o each s ain we e incuba ed a 30◦C,and images
o ep esen a i e samples we e aken as men ioned abo e.
Mic oscopy s udies
Va iable p essu e scanning elec on mic oscopy
These expe imen s we e pe o med on CTT aga pla es ollowing
he me hodology p e iously desc ibed [30] using a FESEM Zeiss
Sup a 40Vp mic oscope (Jena,Ge many) equipped wi h an ene gy-
dispe si e X- ay (EDX) mic oanalysis sys em.
High- esolu ion ansmission elec on mic oscopy
Cells om he c ossing poin and dis al edges o M. xan hus
and S. melilo i om DPAs we e collec ed, ea ed, and analyzed
as p e iously desc ibed [30], using a mic oscope FEI TITAN G2
(Wal ham, MA, USA) equipped wi h a high-angle annula da k
ield (HAADF) ype de ec o and an EDX mic oanalysis sys em.
Fluo escence mic oscopy
To measu e ROS, 100 μl o dichlo o-dihyd o- luo escein diace a e
(DCFH-DA) a 1 μM was ca e ully added o d ops o pu e cul u es
and DPAs o p eda o and p ey a e 48 h o incuba ion. Fluo-
escein was le o ac o 30 min, and images we e aken using
an Olympus IX71 luo escence mic oscope wi h a DP72 digi al
came a. Fluo escence signals we e analyzed using he Olympus
Cell∧Fso wa e.
Resul s
S udies on he ole o i on du ing p eda ion
To add ess he ole o i on du ing he p eda ion o M. xan hus
on S. melilo i, DPAs we e pe o med on media wi h and wi hou
added i on. These esul s showed ha M. xan hus pene a ed
mo e e icien ly in o he p ey colony in media supplemen ed
wi h i on (Fig. 1A), indica ing ha his me al a o s he p eda o y
capabili y o he myxobac e ium. This imp o emen could be
caused by an inc ease in oxida i e s ess in he p ey gene a ed
by he accumula ion o i on. To in es iga e his possibili y, he
amoun o ROS in bo h bac e ia was analyzed using DCFH-DA. The
esul s e ealed ha p eda o s and p ey accumula ed mo e ROS
in cocul u es han in pu e cul u es, al hough his accumula ion
does no appea o be ela ed o he amoun o i on p esen in he
media (Fig. 1A). In addi ion, high- esolu ion ansmission elec on
mic oscopy (HRTEM) wi h EDX mic oanalyses e ealed ha i on
was no accumula ed inside he cells, ega dless o he me al
le els in he medium, and no di e ences in i on dis ibu ion we e
obse ed be ween egions whe e s ains we e alone o in con ac
(Fig. 1B). In con as , phospho us de ec ion was clea ly associa ed
wi h cells. These expe imen s uled ou i on as being esponsible
o he accumula ion o ROS obse ed du ing p eda ion.
To u he in es iga e why i on imp o es p eda ion, gene a ion
imes o p eda o and p ey wild- ype (WT) s ains we e com-
pa ed in media supplemen ed o no wi h he me al, and he
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4|Con e as-Mo eno e al.
Figu e 1. S udies on he ole o i on du ing p eda ion. (A) De e mina ion o ROS accumula ion in M. xan hus and S. melilo i g own in pu e cul u es and
cocul u es.Cells we e incuba ed in CTT medium supplemen ed o no wi h 220 μMFeCl
3. Pic u es we e aken 48 h a e adding DCFH-DA unde a
mic oscope wi h illumina ion om he bo om o using a luo escence il e . The amoun o ROS was measu ed along he whi e ba s in he
luo escence images. In cocul u es, h ee measu es a e shown: M. xan hus Region (Mx), in e ace egion (In e ), and S. melilo i egion (Sm). Expe imen s
we e pe o med in iplica e, and e o numbe s indica e s anda d de ia ions. Two- ailed S uden ’s - es was used o de e mine signi ican di e ences
in ROS accumula ion (∗:P<0.05; ∗∗;P<0.01; ∗∗∗:P<0.001). Compa isons be ween M. xan hus a e depic ed in g een, and be ween S. melilo i in pink. (B)
Dis ance p eda ion assays o M. xan hus e sus S. melilo i we e pe o med in media wi hou o wi h i on supplemen a ion. Cells om a eas whe e each
s ain was alone o a he collision poin we e collec ed a 48 h. images o he same a ea we e ob ained by HRTEM using an HAADF de ec o (uppe
pic u es) o EDX mic oanalysis o i on (middle pic u es) and phospho us (lowe pic u es).
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I on in bac e ial p eda ion |5
esul s showed ha i on does no signi ican ly modi y hese al-
ues (Fig. 2A). Fu he mo e, because M. xan hus mo ili y is equi ed
o p ope p eda ion [16], he expansion a e o he Mx_WT colony
was also measu ed, e ealing ha he diame e o he colony
signi ican ly inc eased in media supplemen ed wi h i on (Fig. 2B).
To ule ou he possibili y ha his inc ease could be he esul o
g ow h, he ne adial expansion o he colony o e ime, which
is equal o he slope o he s aigh line ob ained when g ow h
a e is plo ed agains mo ili y, was de e mined [56]. The esul s
showed ha he mo ili y o Mx_WT inc eased by 12% in media
supplemen ed wi h i on. Al oge he , hese expe imen s indica e
ha oxida i e s ess inc eases du ing p eda o y in e ac ions and
ha i on does no appea o be esponsible o ROS accumula ion.
Howe e , i on imp o es p eda ion, which co ela es wi h a highe
mo ili y a e in M. xan hus.
I on and p eda o and p ey side opho es
accumula e a he p eda o y in e ace in media
wi hou i on supplemen a ion
To ocus on he ole o compe i ion o i on du ing p eda ion, he
amoun o i on a he in e ace whe e bo h bac e ia collided on
CTT medium was de e mined using a iable p essu e scanning
elec on mic oscopy (VPSEM) coupled wi h EDX.Scan mic oanaly-
ses using his echnique e ealed ha whe eas ca bon and oxygen
(indica i e o cell biomass) exhibi ed simila p o iles, oughly
co esponding o he accumula ion o cells in he a ea, i on le els
especially inc eased in hose egions whe e bo h bac e ia con ac ,
ollowing a pa e n di e en om ha desc ibed by he biomass
(Fig. 3A). Because an inc ease in he amoun o me al inside he
cells was no de ec ed (Fig. 1B), hese esul s sugges ha i on
emains ex acellula ly in he p eda o y in e ace.
P e ious indings e ealed ha he M. xan hus and S. melilo i
side opho e biosyn hesis genes (Fig. S3) a e up egula ed du ing
p eda ion (Fig. S4)[40,41]. To moni o his up egula ion in M. xan-
hus,anmxcG-lacZ usion was cons uc ed.The mxcG gene encodes
a non ibosomal pep ide syn he ase in ol ed in he biosyn he-
sis o myxochelins [57,58], which a e side opho es p oduced
by M. xan hus (Fig. S3A). The esul s showed ha he p eda o
inc eased he exp ession o myxochelin biosyn hesis genes when
encoun e ing he p ey in a medium no supplemen ed wi h i on
(Fig. 3B). Simila ly, an S. melilo i s ain ha bo ing an hb-lacZ usion
was cons uc ed. hb genes a e in ol ed in Rz1021 biosyn hesis
(Fig. S3B), he side opho e p oduced by S. melilo i Rm1021 [59].
DPAs con i med ha in medium nonsupplemen ed wi h i on, he
p ey also up egula ed he exp ession o side opho e biosyn hesis
genes (Fig. 3B). As expec ed, he addi ion o i on ep essed he
exp ession o bo h myxochelin and Rz1021 biosyn hesis genes
(Fig. 3B). Al oge he , hese da a indica e ha compe i ion o i on
occu s du ing p eda ion, which migh be decisi e in he ou come
o he in e ac ion. To add ess his ques ion, e o s we e ocused
on s udying he ole o side opho es du ing p eda ion.
P eda o side opho es imp o e p eda ion
To in es iga e he ole o p eda o side opho es, a myxochelin-
de icien mu an (mxcG) was ob ained (Fig. 4A). OPAs we e pe -
o med o quan i y he numbe o cells a e p eda o –p ey in e -
ac ion. When he p eda o y ac i i y o he mxcG mu an was
compa ed wi h ha o Mx_WT, i was obse ed ha , wi hou
i on addi ion, he numbe o hizobial ch omosomes es ima ed
by ddPCR was simila when exposed o he Mx_WT s ain and he
mxcG mu an , whe eas he numbe o M. xan hus ch omosomes
Figu e 2. Gene a ion ime and mo ili y o he WT and mu an s ains
used in his s udy. (A) Doubling ime o myxobac e ial (g een ba s) and
hizobial (pink ba s) s ains g own in CTT medium wi h 220 μMFeCl
3o
wi hou i on supplemen a ion. (B) Expansion o M. xan hus s ains in
CTT medium wi h and wi hou i on addi ion. Diame e o he colonies
was measu ed a e 48 h o incuba ion. Expe imen s we e pe o med in
iplica e, and e o ba s indica e s anda d de ia ions. Two- ailed
S uden ’s - es was used o de e mine signi ican di e ences (∗:P<
0.05; ∗∗:P<0.01; ∗∗∗:P<0.001), which a e shown ollowing his code:
Compa isons o he WT s ains wi h mu an s (g een o M. xan hus and
pink o S. melilo i) g own unde he same condi ions a e depic ed wi h
con inuous lines; compa isons o he M. xan hus mxcG_ u A mu an
e sus he u A mu an g own unde he same condi ions a e depic ed
wi h g een dashed lines; and compa isons be ween he same s ain
g own in media wi h o wi hou i on supplemen a ion a e depic ed as
black con inuous lines.
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6|Con e as-Mo eno e al.
Figu e 3. I on and side opho es accumula e a he p eda o –p ey in e ace. (A) De e mina ion o ca bon, oxygen, and i on a he in e ace
p eda o –p ey by VPSEM wi h EDX mic oanalysis. Only he g aph co esponding o i on is new in his manusc ip . The es o he images we e
ob ained om ou p e ious wo k [30], wi h pe mission. (B) Exp ession o genes in ol ed in myxochelin (le ) and Rz1021 ( igh ) biosyn hesis du ing he
p eda ion o M. xan hus on S. melilo i. S ains ha bo ing he indica ed lacZ usions we e assayed agains he WT s ain o he o he bac e ium in CTT
medium con aining X-gal, wi h o wi hou i on supplemen a ion. Pic u es we e aken a e 48 h o incuba ion wi h illumina ion om he op (le
pic u es) o om he bo om ( igh pic u es). Colo s we e scanned along he lines d awn in he pic u es. To elimina e backg ound om whi e ligh , he
alues shown a e he a io be ween blue and ed (B/R index).
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I on in bac e ial p eda ion |7
Figu e 4. In luence o myxochelin p oduc ion on he p eda o y capabili y o M. xan hus. (A) Analysis o he biosyn hesis o myxochelins in se e al
s ains o M. xan hus. In he uppe pic u es, he Mx_WT s ain and mxcG, u A,andmxcG_ u A mu an s we e cul u ed on blue CAS aga o 72 h. The
yellow halo a ound he colony o igina es om he accumula ion o side opho es. Da a in he lowe pa o he panel show he exp ession le els o
myxochelin biosyn hesis genes in he Mx_WT s ain (mxcG-lacZ)and u A mu an ( u A_mxcG-lacZ). Cells ha bo ing usions be ween myxochelin
biosyn hesis genes and lacZ we e incuba ed on CTT medium wi h o wi hou i on supplemen a ion o 24 h. Th ee samples we e hen collec ed, and
β-galac osidase speci ic ac i i y was quan i ied using o-ni ophenyl galac opy anoside as a subs a e. Speci ic ac i i y is exp essed as nmol o
o-ni ophenol p oduced pe min and mg o p o ein. (B) P eda o y capabili y o he Mx_WT s ain and he mxcG mu an , which does no p oduce
myxochelins (mxcG dele ion), on Sm_WT in media wi h o wi hou i on addi ion. Assays we e pe o med o 72 h in semiquan i a i e o e lapping
p eda ion assays (le pic u es) and quan i ied by ddPCR ( igh g aph). ddPCR expe imen s we e pe o med in iplica e, and e o ba s indica e
s anda d de ia ions. Signi ican di e ences be ween WT s ains and mu an s g own unde he same condi ions we e de e mined using a wo- ailed
S uden ’s - es (∗∗:P<0.01) and a e depic ed wi h con inuous lines. (C) Quali a i e analysis o he p eda o y beha io o a mu an ha o e p oduced
myxochelins ( u A gene is dele ed) in CTT media wi h o wi hou i on addi ion, compa ed wi h he Mx_WT s ain and he mxcG and mxcG_ u A
mu an s. All images in his igu e we e aken a 72 h unde a dissec ing mic oscope wi h illumina ion om he bo om. Ba s ep esen 1 mm unless
o he wise s a ed.
de ec ed in he mxcG mu an was app oxima ely hal o ha o
he Mx_WT s ain (Fig. 4B). Al hough he mxcG mu an exhibi s
a doubling ime and an expansion a e simila o hose o he
Mx_WTs aininpu ecul u es(
Fig. 2), he da a ob ained du ing
p eda ion sugges ha he p ey may in e e e wi h he g ow h o
his mu an . In ac , he exp ession o genes in ol ed in Rz1021
biosyn hesis (de ec ed by using an hb-lacZ s ain) was high e en
when S. melilo i was con on ed wi h he nonside opho e p oduce
mxcG mu an (Fig. S5). In con as , when i on was added o he
medium and side opho e biosyn hesis genes we e no exp essed
in any s ain (Fig. 4A), he numbe o p eda o ch omosomes
(Mx_WT and mxcG mu an ) a e he in e ac ion was simila
(Fig. 4B).
To u he in es iga e he ole o myxochelins in p eda ion,
we decided o cons uc a mu an ha o e p oduced hese
side opho es e en in media supplemen ed wi h i on. The e o e,
i was i s necessa y o iden i y he ep esso ha egula es he
exp ession o myxochelin biosyn hesis in M. xan hus. An analysis
o he genome e ealed ha he gene MXAN_3702 encodes a
p o ein wi h simila i ies o Fu amily membe s. To in es iga e
he unc ion o his p o ein, a s ain ha bo ing a dele ion in his
gene was gene a ed. This mu an o e p oduced side opho es and
exp essed myxochelin biosyn hesis genes a high le els, wi h and
wi hou i on supplemen a ion (Fig. 4A), demons a ing ha his
ep esso , e med Fu A, is he mas e egula o o side opho e
p oduc ion in M. xan hus.The u A mu an exhibi ed a longe
doubling ime and a lowe expansion a e han he Mx_WT (Fig. 2).
The e o e, when he p eda o y capabili y o he u A mu an was
assayed in OPAs,i was ound ha i exhibi ed a diminished abili y
o g ow and kill he p ey (Fig. S6). This esul migh be explained
by he ac ha , in OPAs, he numbe o p eda o cells is ≈100
imes lowe han ha o he p ey (Fig. S2B), which a e unable o
e icien ly g ow in he p esence o a obus Sm_WT wi h a sho e
doubling ime (Fig. 2). To o e come his di icul y and disce n he
ole o myxochelins in p eda ion, DPAs we e pe o med using he
u A mu an as a p eda o . In his case, wi hou i on addi ion, he
a ea o he p ey colony close o he p eda o was clea e , e en
be o e he p eda o had eached i , compa ed wi h he egion a
om he mu an (Fig. 4C). This obse a ion can be explained by
conside ing ha he mu an o e p oduces myxochelins (Fig. 4A),
which can di use and each he p ey colony in ad ance,deple ing
i on o he p ey and inhibi ing i s g ow h. In con as , in CTT wi h
i on,in which his me al is no limi ing o p ey, he beha io o he
u A mu an and Mx_WT s ain appea ed o be simila (Fig. 4C).
Hence, because he u A mu an , in addi ion o o e p oducing
myxochelins, exhibi s o he pheno ypic de ec s, a double mu an
mxcG_ u A (in which myxochelin p oduc ion was abolished) was
gene a ed and analyzed o con i m he ole o side opho es in
p eda ion (Fig. 4A). This mu an e ained de ec s in g ow h a e
and mo ili y (Fig. 2). The esul s in DPAs e ealed ha his double
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8|Con e as-Mo eno e al.
Figu e 5. In luence o Rz1021 p oduc ion on he de ensi e capabili y o S. melilo i agains p eda ion. (A) Analysis o Rz1021 p oduc ion in he hizobial
s ains used in his s udy. In he uppe images, hizobial s ains we e cul u ed on blue CAS aga o 48 h. The yellow halo a ound he colony esul s
om he accumula ion o side opho es. Da a in he lowe pa o he panel show he exp ession le els o Rz1021 biosyn hesis genes in he Sm_WT
s ain ( hb-lacZ)and i A and h A mu an s ( i A_ hb-lacZ and h A_ hb-lacZ, espec i ely). Cells ha bo ing usions be ween Rz1021 biosyn hesis genes
and lacZ we e incuba ed in CTT medium wi h o wi hou i on supplemen a ion o 24 h. Th ee samples we e hen collec ed, and β-galac osidase
speci ic ac i i y was quan i ied using o-ni ophenyl galac opy anoside as a subs a e. Speci ic ac i i y is exp essed as nmol o o-ni ophenol p oduced
pe min and mg o p o ein. (B) P eda o y beha io o he Mx_WT s ain agains he Sm_WT and mu an s ains ha do no p oduce ( hbA and
hbA_ i A mu an s) o o e p oduce ( h A and i A mu an s) Rz1021 was de e mined a 72 h o incuba ion by semiquan i a i e analysis o he
cocul u es wi h o wi hou i on supplemen a ion (le pic u es). Pic u es we e aken wi h illumina ion om he bo om. Quan i ica ion was pe o med
by ddPCR a e 72 h o in e ac ion ( igh g aph). ddPCR expe imen s we e pe o med in iplica e, and e o ba s indica e s anda d de ia ions.
Two- ailed S uden ’s - es was used o de e mine signi ican di e ences (∗:P<0.05; ∗∗:P<0.01; ∗∗∗:P<0.001), which a e shown ollowing his code:
Compa isons o he WT s ains wi h mu an s (g een o M. xan hus and pink o S. melilo i) g own unde he same condi ions a e depic ed wi h
con inuous lines ( op); compa isons o he same s ain in he same in e ac ion g own wi h and wi hou i on a e also depic ed wi h con inuous lines
(bo om); compa isons o Mx_WT e sus i A and hbA_ i A mu an s g own unde he same condi ions a e depic ed wi h dashed lines (g een o M.
xan hus and pink o S. melilo i).
mu an does no inhibi g ow h o he p ey be o e eaching i
as he u A mu an does (Fig. 4C), indica ing ha his inhibi ion
o g ow h obse ed in he p ey by he u A mu an is caused
by myxochelins. The e o e, he esul s ob ained wi h M. xan hus
mu an s (nonp oduce s and o e p oduce s o myxochelins)
indica e ha p eda o side opho es dec ease i on a ailabili y o
p ey and acili a e p eda ion.
P ey side opho es con ibu e o de ense agains
p eda o a ack
The ole o p ey side opho es du ing p eda ion was also ana-
lyzed by compa ing he beha io o Sm_WT wi h ha o mu an s
al e ed in he p oduc ion and up ake o Rz1021 (Fig. 5A)[60].
P eda ion expe imen s pe o med wi h he nonRz1021 p oduce
hbA mu an e ealed educed su i al o he p ey o p eda o y
a ack in i on-limi ed media bu no in media supplemen ed wi h
he me al (Fig. 5B). As his mu an does no exhibi a lowe g ow h
a e han he Sm_WT s ain (Fig. 2A), he dec ease o su i al
o he hbA mu an indica es ha i on acquisi ion acili a ed by
Rz1021 con ibu es o p ey esis ance o p eda o y a ack.
To in es iga e he ole o side opho es in p ey esis ance, a
s ain ha o e p oduced Rz1021 was gene a ed by dele ion o he
i A gene, which encodes he ep esso o Rz1021 biosyn hesis
genes [61].The i A mu an o e p oduced side opho es,exp essed
Rz1021 biosyn hesis genes ega dless o he i on le els in he
medium (Fig. 5A), and exhibi ed a longe doubling ime han
Sm_WT (Fig. 2A). In media wi hou i on supplemen a ion, he
i A mu an esis ed p eda ion (Fig. 5B). Mo eo e , he numbe o
Mx_WT ch omosomes when con on ed wi h he i A mu an was
lowe ha when exposed o Sm_WT, e en in media supplemen ed
wi h i on (Fig. 5B), indica ing ha M. xan hus has di icul ies g ow-
ing when Rz1021 is o e p oduced. No e ha M. xan hus up egu-
la ed myxochelin biosyn hesis genes du ing in e ac ion wi h he
i A mu an , e en in CTT supplemen ed wi h i on (Fig. S7). Hence,
because he i A mu an up egula es he exp ession o se e al
o he genes in addi ion o hose in ol ed in he biosyn hesis o
side opho es [61], a double mu an hbA_ i A was cons uc ed
(in which side opho e p oduc ion was abolished) o con i m he
ole o Rz1021 in p eda ion (Fig. 5A). The esul s showed ha his
double mu an was mo e sensi i e o p eda ion han he i A
mu an (Fig. 5B), con i ming ha he esis ance o p eda ion o
he i A mu an is caused by Rz1021 o e p oduc ion.
To co obo a e he ole o Rz1021, he gene coding o he TBDT
Rh A, which is equi ed o anspo ing Fe-Rz1021 h ough he
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I on in bac e ial p eda ion |9
ou e memb ane [59], was inac i a ed. The h A mu an o e ex-
p essed Rz1021 biosyn hesis genes only when i on was no added
(Fig. 5A) because i was unable o ake up e iside opho es. In
con as o he i A mu an , he doubling ime o he h A mu an
was simila o ha o he Sm_WT (Fig. 2A). P eda ion assays
e ealed ha he h A mu an was mo e esis an o Mx_WT
p eda ion han he Sm_WT in media no supplemen ed wi h i on
(Fig. 5B). Howe e , when i on was added o he media and Rz1021
biosyn hesis genes we e no exp essed (Fig. 5A), su i al le els o
he h A mu an and he Sm_WT we e simila (Fig. 5B),con i ming
ha Rz1021 p o ec s S. melilo i om p eda ion by deple ing he
i on supply om he p eda o .
P eda o y capabili y o p eda o mu an s agains
p ey mu an s
To u he es he ole o side opho es, p eda o mu an s we e
analyzed o hei p eda o y beha io agains p ey mu an s.
The esul s e ealed ha he mxcG mu an , a nonp oduce o
myxochelins, could no g ow p ope ly when con on ed wi h
hizobia ha o e p oduced Rz1021 ( i A and h A mu an s in
media wi hou added i on, and only i A mu an in media wi h
i on supplemen a ion) (Fig. 6A). When mxcG was assayed agains
mu an s nonp oduce s o Rz1021 ( hbA and hbA_ i A), he
numbe o p eda o ch omosomes ob ained (Fig. 6A) was simila
o ha obse ed in he Mx_WT (Fig. 5A).
When p eda ion was ca ied ou by he u A mu an , which
always o e exp essed myxochelin biosyn hesis genes (Fig. 4A),
quali a i e analyses in DPAs e ealed ha , in media no supple-
men ed wi h i on, his mu an pene a ed he hizobial colonies
o nonp oduce mu an s o Rz1021 ( hbA and hbA_ i A mu an s)
a a simila a e han in colonies o he Sm_WT (Fig. 6B). How-
e e , unde he same condi ions, he u A mu an could no e i-
cien ly pene a e he h A and i A mu an colonies (Fig. 6B).These
esul s co ela e wi h he amoun o Rz1021 p oduced by hese
wo hizobial mu an s (Fig. 5A). When he same mu an s we e
assayed in i on-en iched media, he u A mu an pene a ed e y
e icien ly he colonies o hbA, hbA_ i A,and h A mu an s,bu
no in he i A mu an colony (Fig. 6B), co esponding o he ac
ha hbA, hbA_ i A,and h A mu an s do no syn hesize Rz1021
unde hese condi ions, whe eas he i A mu an does (Fig. 5A).
The e iciency o he u A mu an o pene a e he hizobial
colonies is he esul o myxochelin o e p oduc ion, as indica ed
by he educed abili y o pene a e he hizobial colonies exhibi ed
by he myxochelin-de icien mxcG_ u A double mu an (Fig. 6C).
These da a con i m ha compe i ion o i on is a cen al ba le-
ield be ween p eda o and p ey and ha side opho es al e he
ou come o p eda o –p ey in e ac ions.
Discussion
Myxobac e ial p eda ion is a mul i ac o ial p ocess in which cells
use a as a senal o hun , kill, lyse, and consume p ey [62], which
a ies om one p ey o ano he [20,36,40,63,64]. Wi hin his
a senal, me als a e eme ging as no el weapons o kill p ey [30,36,
39]. In his s udy, we ocused on he ole o i on and side opho es
du ing he p eda o y in e ac ion o M. xan hus wi h S. melilo i.
S udies ha e e ealed ha in oxica ion wi h i on is no a
majo mechanism used by he p eda o because he me al is
no accumula ed inside he cells and he accumula ion o ROS
in cells du ing p eda ion is simila ega dless o he amoun o
me al included in he media (Fig. 1). Mo eo e , ansc ip omic
s udies du ing he p eda ion o M. xan hus on S. melilo i ha e
shown ha genes in ol ed in he de oxi ica ion o ROS a e no
up egula ed in he p ey, excep o supe oxide dismu ase [41].
Simila ly, no gene up egula ed du ing p eda ion in he p eda o
appea s o play a ole in i on in oxica ion o he p ey [40].Howe e ,
i on imp o es pene a ion o he p eda o in o he p ey colony,
because he expansion a e o he p eda o inc eases wi h i on,
which undoub edly a o s eaching he p ey and es ablishing
close con ac wi h he cells. Ne e heless, i canno be uled ou
ha se e al o he mechanisms used by he p eda o may be mo e
e icien in i on-en iched media.
In con as o in oxica ion wi h i on, compe i ion o his me al
appea s o be decisi e in he ou come o he p eda o –p ey in e -
ac ion. Da a ob ained in his wo k wi h mu an s ha do no p o-
duce o o e p oduce side opho es demons a e ha he compe i-
o ha is able o deple e he i on supply o he i al will p e ail.
O he s udies also poin in his di ec ion. Fo ins ance, an M. xan-
hus mu an in MXAN_6911, which is a pu a i e e imyxochelin
anspo e , exhibi s less e icien p eda ion on P. ae uginosa [39].
Howe e , his mu a ion has no e ec on in acellula i on le els
o side opho e syn hesis, p obably because he e a e wo ypes
o myxochelins, A and B, which could be anspo ed by di e en
TBDTs. Th ee genes ha encode pu a i e TBDTs (MXAN_1316,
MXAN_5023, and MXAN_6911) a e up egula ed du ing p eda ion
on S. melilo i (Fig. S4A), wi h he ups eam egions o MXAN_5023
and MXAN_6911 exhibi ing a Fu box [40]. In addi ion, a p e ious
s udy showed ha mu an s in componen s o he ABC ans-
po e ha in oduces e imyxochelins in o he M. xan hus cy o-
plasm a e de ec i e in p eda ion [39]. This s udy also gene a ed a
mu an ha p oduced ewe side opho es han he Mx_WT s ain,
which was also de ec i e in p eda ion [39]. Howe e , he pa hway
in ol ed in myxochelin A and B biosyn hesis om cho isma e,
depic ed in Fig. S3A, has been well es ablished [57,58], and he
gene ha hese au ho s mu a ed (MXAN_3618), al hough encod-
ing a non ibosomal pep ide syn he ase, has no been epo ed o
be in ol ed in his p ocess. The e o e, i canno be uled ou ha
o he p ocesses, in addi ion o myxochelin biosyn hesis, can be
impai ed in his mu an , which may also be in ol ed in p eda ion.
Se e al ansc ip omes using M. xan hus as a p eda o agains
di e se p ey, such as S. coelicolo [36], E. coli,andM. lu eus [42],
ha e been published, and in all o hem, side opho e biosyn hesis
genes we e up egula ed in bo h p eda o and p ey.Mo eo e ,using
ano he myxobac e ium as a p eda o (C. e ugineus) agains P.
pu ida, he same esul was ob ained [38], which indica es ha
compe i ion o i on may be a gene al mechanism ha pa ici-
pa es in myxobac e ial p eda ion.These ansc ip omes ha e also
e ealed ha p eda ion is a dynamic p ocess, whe e he p o iles
o p eda o and p ey genes di e en ially exp essed a ea lie and
la e imes o he in e ac ion a e no iden ical, deno ing ha each
bac e ium adap s o he esponse o he o he . In he case o he
p eda ion o M. xan hus on S. coelicolo , he deple ion o i on igge s
he biosyn hesis o he an ibio ic ac ino hodin in he p ey [36],
indica ing ha compe i ion o his me al unc ions as a i s line
o a ack-de ense du ing he in e ac ion.
Two me als ha e been epo ed o be used by M. xan hus du ing
i s p eda o y in e ac ion wi h S. melilo i: coppe and i on. Coppe
appea s o be used o kill p ey by oxida i e s ess because he
me al accumula es inside he cells, he p eda o up egula es he
exp ession o genes in ol ed in coppe de oxi ica ion, and he
p ey esponds by syn hesizing melanin o p o ec om oxida i e
s ess [30]. In he case o i on, compe i ion o he me al appea s
o be mo e ele an han in oxica ion.
A pH 7,i on has a solubili y o 1.4 ×10−9M[65],which makes i
e y inaccessible o o ganisms. Mo eo e , many me alloenzymes
equi e i on as a co ac o [66,67]. To o e come his p oblem,
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