Global coppe esponse o he soil bac e ial p eda o Myxococcus xan hus
and i s con ibu ion o an ibio ic c oss- esis ance
F ancisco Ja ie Ma cos-To es , Juana P´
e ez, Da id To ens-Gonz´
alez,
Miguel ´
Angel Ga cía-Ped osa, F ancisco Ja ie Con e as-Mo eno, Au elio Mo aleda-Mu˜
noz
*
Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada, G anada 18071, Spain
ARTICLE INFO
Keywo ds:
Coppe homeos asis
An ibio ic co- esis ance
Bac e ial p eda ion
Myxococcus xan hus
Sino hizobium melilo i
ABSTRACT
Coppe accumula ion in ag icul u al soils poses en i onmen al challenges by selec ing coppe - esis an bac e ia
and also con ibu ing o he co-selec ion o an ibio ic- esis an bac e ia. In addi ion, coppe in luences bac e ial
p eda o -p ey in e ac ions, po en ially al e ing mic obial ecosys ems. Myxococcus xan hus, a soil-dwelling bac-
e ium, p eys on o he mic oo ganisms, including Sino hizobium melilo i, a symbio ic ni ogen- ixing bac e ium
associa ed wi h leguminous plan s. The ole o coppe in M. xan hus in e ac ions emains poo ly unde s ood,
al hough i accumula es a he p eda o -p ey in e ace. In his s udy, we explo e he ansc ip omic esponse o
M. xan hus o coppe s ess in bo h monocul u es and co-cul u es wi h S. melilo i. Ou analysis iden i ied many
myxobac e ial coppe - egula ed ansc ip s, and s udies on mu an s ains in some coppe -induced genes
e ealed he ole o wo e lux pumps in c oss- esis ance o coppe and e acyclines. These indings p o ide new
insigh s in o he adap i e mechanisms o M. xan hus in esponse o coppe , wi h implica ions o he co-selec ion
o an ibio ic esis ance and he b oade impac o coppe on mic obial communi y dynamics in soil ecosys ems.
1. In oduc ion
Myxococcus xan hus is a soil bac e ium om he phylum Myx-
ococco a, well-known o i s complex mul icellula li e cycle. In he
absence o nu ien s, M. xan hus unde goes a unique de elopmen al
p ocess in which indi idual cells agg ega e and o m mac oscopic,
ui ing bodies, which ep esen a key aspec o i s su i al s a egies.
These ui ing bodies a e o med om h ee dis inc cell subpopula ions
ha di e en ia e and di ide labo , exempli ying mul icellula beha io
among bac e ia (Mu˜
noz-Do ado e al., 2016, 2019; Ma cos-To es e al.,
2020). In he p esence o o he mic oo ganisms, M. xan hus beha es as a
coope a i e epibio ic p eda o (P´
e ez e al., 2016). The combina ion o
seconda y me aboli es, sec e ed enzymes and con ac -dependen killing
app oaches ensu es ha M. xan hus can success ully p ey on a wide
ange o o ganisms and u ilize hei deg ada ion p oduc s o i s own
g ow h and su i al (See e al., 2021; Thie y e al., 2022;
Con e as-Mo eno e al., 2024b; He ou e al., 2024). To e icien ly
na iga e i s en i onmen , ei he in sea ch o p ey o when o ming
ui ing bodies, M. xan hus u ilizes wo dis inc mo ili y sys ems:
ad en u ous mo ili y (A-mo ili y), which is associa ed wi h indi idual
cell mo emen and social mo ili y (S-mo ili y), which go e ns g oup o
coo dina ed displacemen . Bo h mo ili y sys ems ha e been shown o be
equi ed o e icien p eda ion (P´
e ez e al., 2014). Fu he mo e, an-
sc ip omic analyses ha e e idenced ha du ing p eda ion, M. xan hus
up egula es genes ela ed o killing, lysing, and consuming p ey, while i
down egula es genes in ol ed in he de elopmen al cycle (P´
e ez e al.,
2022), which sugges s a shi in me abolic and egula o y p io i ies,
allowing M. xan hus o maximize i s p eda o y e iciency.
Se e al s udies ha e indica ed ha me als commonly p esen in he
soil en i onmen play signi ican oles in modula ing p eda o y in-
e ac ions. Thus, while i on is well-s udied o i s e ec s on mic obial
compe i ion and p eda ion (Lee e al., 2020; Con e as-Mo eno e al.,
2024b), he ole o coppe emains mo e elusi e, al hough p e ious
s udies ha e epo ed he accumula ion o coppe inside he phagosome
o p eda o y p o ozoans o kill bac e ia (Ge man e al., 2013; Hao e al.,
2016), as well as he pa icipa ion o his me al in he p eda o y ac i i y
o he bac e ia Cup ia idus neca o (Secca eccia e al., 2016) and
M. xan hus (Con e as-Mo eno e al., 2020, 2024a). Du ing he in e ac-
ion o M. xan hus wi h Sino hizobium melilo i, coppe accumula es a he
p eda o -p ey in e ace and, al hough i s ole in his in e ac ion is no
ully unde s ood, his coppe accumula ion induces melanin p oduc ion
in S. melilo i (Con e as-Mo eno e al., 2020).
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (A. Mo aleda-Mu˜
noz).
Con en s lis s a ailable a ScienceDi ec
Mic obiological Resea ch
jou nal homepage: www.else ie .com/loca e/mic es
h ps://doi.o g/10.1016/j.mic es.2025.128357
Recei ed 29 July 2025; Recei ed in e ised o m 22 Sep embe 2025; Accep ed 29 Sep embe 2025
Mic obiological Resea ch 302 (2026) 128357
A ailable online 1 Oc obe 2025
0944-5013/© 2025 The Au ho (s). Published by Else ie GmbH. This is an open access a icle unde he CC BY license
(
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
Coppe is an essen ial mic onu ien wi h impo an biological
unc ions, since i s edox ac i i y makes i bo h a c i ical co ac o o
cup oenzymes and a po en ial oxin when p esen in excess (Fes a and
Thiele, 2011). Consequen ly, bac e ia, like all li ing o ganisms, ha e
e ol ed sophis ica ed mechanisms o manage he dual e ec s o coppe .
To main ain coppe homeos asis, bac e ia u ilize a ious s a egies,
including coppe -e lux pumps and de oxi ica ion sys ems, such as
p o eins ha can con e oxic Cu⁺ back o less eac i e Cu
2+
, and
coppe -binding p o eins and chape ones ha seques e coppe o p e-
en i om in e ac ing wi h sensi i e cellula a ge s. Despi e hese
p o ec i e mechanisms, he ha m ul oxida ion p oduced by coppe s ill
induces he cell epai sys ems (Mo aleda-Mu˜
noz e al., 2005; Rensing
and McDe i , 2013; P´
e ez e al., 2018).
The coppe esponse in M. xan hus in ol es a sophis ica ed egula-
o y ne wo k ha comp ises many genes, he majo i y o which a e
clus e ed in o wo dis inc ch omosomal egions, coppe egion 1 (CR1)
and coppe egion 2 (CR2) (Mo aleda-Mu˜
noz e al., 2010a, 2010b; P´
e ez
e al., 2018). CR1 includes genes encoding o wo HME-RND hea y
me al e lux sys ems ha a e induced by coppe , while CR2 houses many
genes ela ed o coppe homeos asis ha a e unde he con ol o wo
egula o y elemen s: he wo-componen sys em (TCS) Co SR and he
coppe -dependen ex acy oplasmic unc ion (ECF) sigma ac o Co E
(Fig. S1A). The his idine kinase Co S de ec s Cu
2+
in he pe iplasmic
space and, along wi h he esponse egula o Co R, modula es a
nine-gene ope on in ol ed in he main enance esponse o coppe
(S´
anchez-Su il e al., 2013, 2016). Co E senses he coppe edox s a e in
he cy oplasm and Co E-dependen genes a e ac i a ed quickly upon
exposu e o coppe (G´
omez-San os e al., 2011), ini ia ing an immedia e
de oxi ica ion esponse. This phased esponse helps M. xan hus o
main ain coppe homeos asis unde a ying le els o coppe s ess. In
addi ion o he genes loca ed in CR1 and CR2, he M. xan hus genome
also con ains o he genes encoding HME-RND sys ems, which a e
induced by coppe (Mo aleda-Mu˜
noz e al., 2010a).
Unde s anding how M. xan hus manages coppe a he molecula
le el du ing p eda ion could p o ide aluable insigh s in o bac e ial
su i al s a egies and me al homeos asis du ing p eda o y in e ac ions.
This complex esponse is c i ical o su i e in i s na u al habi a , he soil,
whe e besides na u al coppe luc ua ions, he concen a ion o his
me al is inc easing due o human ac i i ies, pa icula ly in ag icul u al
a eas because o i s use as a biocide. Coppe accumula ion in soils has
been shown o signi ican ly al e mic obial communi ies and ac as a
selec i e p essu e a o ing he p e alence o ce ain genes, such as
an ibio ic esis ance genes (ARGs), in mic obial popula ions (Ballabio
e al., 2018; Pan e al., 2023). In ac , a g owing numbe o s udies ha e
demons a ed ha he p opaga ion o ARGs in di e en en i onmen s is
d i en by he p esence o me als (Pal e al., 2017; Zhao e al., 2024). In
pa icula , coppe pollu ion has been shown o p omo e he de elop-
men and p opaga ion o an ibio ic esis ance, mainly by co-selec ion
phenomena and by ho izon al gene ans e (HGT) o esis ance genes
(Pal e al., 2017; Ede e al., 2023). Co-selec ion occu s when he p es-
ence o coppe exe s a selec i e p essu e on mic obial communi ies,
leading o he simul aneous selec ion o me al esis ance genes and
ARGs. This p ocess can a ise h ough h ee p ima y mechanisms:
co- esis ance ( esis ance genes o me als and an ibio ics a e physically
loca ed oge he on he same mobile gene ic elemen ), c oss- esis ance
( esis ance genes can con e esis ance o bo h me als and an ibio ics),
and co- egula ion (a egula o y elemen can simul aneously egula e he
exp ession o genes o me als and an ibio ics esis ance).
Mo eo e , coppe pollu ion can also accele a e HGT o ARGs, u he
acili a ing he dissemina ion o ARGs ac oss di e en bac e ial species
(Va s e al., 2022). Fu he mo e, HGT can be acili a ed by mic obial
in e plays such as p eda o -p ey in e ac ions. M. xan hus p ey on a b oad
a ay o o ganisms, including clinically ele an pa hogens, pa hogenic
bac e ia in bio ilms and soil bac e ia (P´
e ez e al., 2011, 2014; Li ing-
s one e al., 2017; Kamada e al., 2023; A akal e al., 2025), which could
ac as a dono o genes ha could change p ey suscep ibili y o
an ibio ics. In ac , a ecen s udy epo ed he exchange o ARGs ac oss
membe s o he Myxococco a and Pseudomonado a phyla (B own e al.,
2024). Mo eo e , some e idence poin o a co ela ion be ween he
p esence o M. xan hus in na u al soil communi ies and he abundance o
an ibio ic- esis an bac e ia (Saha e al., 2025). As desc ibed abo e,
M. xan hus has many coppe - esponsi e genes in ol ed in me al ho-
meos asis, and hei abundance could con ibu e o coppe and an i-
bio ic co-selec ion by c oss- esis ance o co- egula ion e en s.
In his s udy, we del ed in o he global esponse o coppe in
M. xan hus monocul u es and du ing p eda ion, and de e mined which
coppe - esis ance genes o his bac e ium con ibu e o bac e ial esis-
ance o an ibio ics by co-selec ion. These esul s no only expand ou
knowledge o he coppe esponse in soil bac e ia bu also shed ligh on
he ole o his me al in bac e ial p eda ion and iden i y new genes
in ol ed in me al-induced an ibio ic co-selec ion ha can help o com-
ba an ibio ic esis ance, one o he bigges heal h challenges o he XXI
cen u y.
2. Ma e ials and Me hods
2.1. Bac e ial s ains, plasmids, and cul u e condi ions
Bac e ial s ains and plasmids used in his wo k a e lis ed in Table S1.
M. xan hus and S. melilo i s ains we e g own in CTT and TY b o h,
espec i ely, as p e iously desc ibed (P´
e ez e al., 2022) wi h igo ous
shaking a 30◦C. CTT aga pla es con aining Di co aga (Bec on Dick-
inson, Spa ks, MD, Uni ed S a es) we e supplemen ed wi h kanamycin
(80 µg/ml) o galac ose (10 mg/ml), when necessa y.
2.2. Cons uc ion o he in- ame dele ion mu an s
Since p e ious s udies o M. xan hus coppe homeos asis we e pe -
o med on he DZF1 s ain, in his wo k we ob ained in- ame dele ion
mu an s in he DK1622 backg ound using he p e iously epo ed
me hodology (Ma cos-To es e al., 2016). B ie ly, plasmids lis ed in
Table S1 we e in oduced in o M. xan hus DK1622 by elec opo a ion o
ob ain in eg a ion in o he ch omosome by homologous ecombina ion.
Kanamycin- esis an me odiploid s ains we e g own on 1 % galac ose
CTT aga pla es wi hou kanamycin o acili a e loss o he plasmid
h ough a second homologous ecombina ion. Kanamycin-sensi i e and
galac ose- esis an s ains we e analyzed by Sou he n blo ing o check
o in- ame dele ions.
2.3. Pheno ypic cha ac e iza ion o he mu an s ains
Each pheno ypical analysis has been pe o med a leas wo imes o
ensu e he soundness o he ou comes. To analyze he esponse o he
coppe limi a ion o he ΔcopC s ain, we moni o ed he OD
600
o h ee
eplica es o he wild- ype (WT) and mu an s ains inocula ed a an
ini ial op ical densi y a 600 nm (OD
600
) o 0.05 in he p esence o
di e en concen a ions o e a hiomolybda e (TTM), a Cu
2+
-speci ic
chela o , a e 24 h o g ow h. Simila ly, coppe esis ance was assayed
o he Δczc2 s ain by analyzing he g ow h o he WT and mu an
s ains in he p esence o di e en concen a ions o CuSO
4
. Since
M. xan hus has been epo ed o exhibi di e en coppe esis ance
capabili y when p e-incuba ed wi h coppe (S´
anchez-Su il e al., 2013),
g ow h was also es ed wi h cells p e-adap ed o 300
μ
M CuSO
4
.
Mo ili y o he Δczc2 s ain was es ed by acking he colony
diame e o h ee eplica es o 10
μ
l d ops a OD
600
o 15 o he WT and
mu an s ains in CTT aga pla es wi h di e en concen a ions o
CuSO
4
. As p e iously desc ibed, ad en u ous mo ili y was assayed in
CTT pla es wi h 1.5 % aga , while social mo ili y was moni o ed in CTT
pla es wi h 0.5 % aga (G´
omez-San os e al., 2012). The colony diame e
was measu ed e e y 24 h o 120 h and used o calcula e he colony
expansion a es o each s ain.
P eda o y assays o s udy he in e ac ion be ween M. xan hus and
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
2
S. melilo i we e pe o med as p e iously desc ibed (Con e as-Mo eno
e al., 2020), using cul u es a OD
600
o 15 and 5, espec i ely, in media
wi h and wi hou 900 µM CuSO
4
supplemen a ion. Pla es om 8 epli-
ca es o each condi ion we e incuba ed a 30ºC and pic u es om
ep esen a i e samples we e aken.
A modi ied e sion o he Ki by-Baue disk di usion suscep ibili y
me hod was used o assess an ibio ic esis ance using h ee eplica es.
B ie ly, 2 ml o M. xan hus liquid cul u es a OD
600
o 0.3 we e mixed
wi h 5 ml o CTT so aga (0.8 % aga ) and pou ed on CTT aga pla es
wi h o wi hou 300
μ
M CuSO
4
(o 50
μ
M CuSO
4
o he assays wi h he
coppe -sensi i e Δcus2 s ain). A e solidi ica ion o he o e lays, 6 mm
an ibio ic disks (Oxoid Holdings L d, The mo Fishe Scien i ic) we e
placed on op, and he pla es we e incuba ed a 30ºC o 72 h be o e
measu ing he diame e o he inhibi ion halo. The an ibio ics es ed and
he amoun s o each one a e collec ed in Table S2.
In addi ion, a minimum inhibi o y concen a ion (MIC) assay was
pe o med using h ee eplica es o de e mine he suscep ibili y o WT
and Δcus3 s ains o nalidixic acid. B ie ly, he myxobac e ial s ains
we e g own o e nigh in CTT medium and dilu ed a OD
600
o 0.05 in
CTT medium supplemen ed wi h 300 µM CuSO
4
and 0, 25, 50, 100, 200,
400, o 800 µg/ml. Bac e ial g ow h was assessed o each an ibio ic
concen a ion a e an incuba ion pe iod o 24 h a 30◦C and he MIC
alue o nalidixic acid was iden i ied as he lowes concen a ion o he
an imic obial equi ed o inhibi isible g ow h o he es ed s ains.
2.4. P epa a ion o samples o RNA-Seq
To ob ain he samples o ansc ip omic analysis, M. xan hus
DK1622 and S. melilo i Rm1021 WT s ains we e g own o OD
600
o 1.
Then, he cul u es we e concen a ed in TM bu e (10 mM T is–HCl [pH
7.6], 1 mM MgSO
4
) o a inal OD
600
o 15. Fo each o he wo eplica es
o he co-cul u e samples, h ee 10-µl d ops o he hizobial suspension
we e deposi ed on he su ace o CTT aga pla es wi h and wi hou
300 µM CuSO
4
supplemen a ion and allowed o d y. This me al con-
cen a ion was selec ed as i does no a ec physiological p ocesses, such
as cell iabili y o mo ili y, du ing he g ow h o he DK1622 M. xan hus
WT s ain (G´
omez-San os e al., 2012), bu induces he exp ession o
coppe - esponsi e genes (Mo aleda-Mu˜
noz e al., 2010a, 2010b). Nex ,
10-µl d ops o he M. xan hus suspension we e deposi ed on op o each
o he hizobial colonies o a subse o he pla es o ob ain samples o
p eda o y and p ey in e ac ing cells (samples Mx_Sm and Mx_Sm_Cu),
while o he monocul u e samples, ano he subse o samples o
M. xan hus we e kep g owing alone (samples Mx and Mx_Cu). A e 2, 6,
and 10 h o incuba ion, wo eplica es om each o he ou condi ions
we e ha es ed om he pla es. Pelle s om each sample we e esus-
pended immedia ely in o 0.5 ml o RNA P o ec Bac e ia Reagen (Qia-
gen, Hilden, Ge many), incuba ed a oom empe a u e o 5 min, and
ha es ed by cen i uga ion a 5000x g o 10 min (4◦C). Nex , a e
emo al o he supe na an , he pelle s we e s o ed a −80◦C.
2.5. RNA ex ac ion
To pu i y RNA, he ozen pelle s we e hawed, and he cells we e
lysed o 10 min a oom empe a u e wi h 250
μ
l o 3 mg/ml lysozyme
(Roche Diagnos ic, Mannheim, Ge many) and 0.4 mg/ml p o einase K
(Ambion, Ca lsbad, CA, Uni ed S a es) p epa ed in TE bu e (10 mM
T is-HCl; 1 mM e hylenediamine e aace ic acid [EDTA], pH 8.0).
RNeasy Mini Ki (Qiagen, Hilden, Ge many) was used o RNA ex ac-
ion, pe o ming on-column DNase diges ion wi h he RNAse- ee DNase
se (Qiagen, Hilden, Ge many), elu ing each sample in 50 µl o RNase-
ee wa e .
2.6. Lib a y p epa a ion, sequencing, and ansc ip omic da a analysis
To al RNA samples we e p ocessed by No ogene [No ogene Eu ope,
Camb idge, Uni ed Kingdom], including RNA deple ion om o al RNA
samples wi h he Illumina Ribo-Ze o Plus RNA Reduc ion Ki (Illumina,
Inc.). The emaining RNA was p ocessed acco ding o he p ocedu es
desc ibed in P´
e ez e al. (2022) p o iding, on a e age, 23.53 million aw
eads and a genome co e age o 386.58x. This co e age d opped o
380.50x a e aw eads il e ing, which implies emo ing eads wi h
adap o con amina ion, wi h mo e han 10 % o unce ain nucleo ides
and/o wi h mo e han 50 % o low-quali y nucleo ides. FPKM ( ag-
men s pe kilobase o ansc ip pe million agmen s mapped)
no maliza ion was used o compa ison o samples (Table S3). The
a e age FPKM alues o he wo eplica es we e used o calcula e he
Log2 old change (Log2FC) and hose genes wi h a padj<0.05 we e
conside ed di e en ially exp essed genes (DEGs) (Table S4).
2.7. S a is ical analysis
G aphical da a a e exp essed as he a e age ±s anda d de ia ion o
h ee independen eplica es. S a is ical e alua ions we e conduc ed
using G aphPap P ism 8.0.2. (G aphPad So wa e Inc.), and s a is ical
signi icance be ween g oups was assessed using an independen wo-
ailed S uden ’s - es , wi h a P- alue o <0.05 indica ing a signi ican
di e ence. G aphical and hea map ep esen a ions we e c ea ed using
Mic oso ® Excel® o Mic oso 365 MSO .2506.
2.8. Da a a ailabili y
T ansc ip ome sequencing da a ( aw- eads) can be ound a
h ps://www.ncbi.nlm.nih.go / unde he Biop ojec accession numbe
PRJNA1164694.
3. Resul s and discussion
3.1. M. xan hus coppe esponse in monocul u es
Coppe esponse in M. xan hus was analyzed by compa ing an-
sc ip s a e cul u e in he absence and p esence o 300
μ
M CuSO
4
a
h ee di e en ime poin s (2, 6, and 10 h). In ou s udies, all DEGs wi h |
Log2FC| >1 (Table S4) we e manually analyzed.
3.1.1. Up egula ed genes in he p esence o coppe a e mos ly in ol ed in
coppe e lux and de oxi ica ion
Among he mos up egula ed DEGs a e hose in CR1 and CR2, as well
as he HME-RND e lux sys ems p e iously epo ed as being in ol ed in
coppe homeos asis (Mo aleda-Mu˜
noz e al., 2010a, 2010b) (Fig. S1A
and B; Table S4).
CR2 includes he Co E- egula ed genes encoding he mul icoppe
oxidase (MCO) CuoB, he P
1B
- ype ATPase CopB, an ou e memb ane
p o ein OmpB, and a p o ein wi h a hea y me al-associa ed domain
(MXAN_3427) (Fig. S1A). T ansc ip omic da a indica e ha genes in he
Co E egulon show an exp ession pa e n ypical o he immedia e
esponse o coppe , exhibi ing an ea ly peak o exp ession ha d ops a
la e imes (Fig. 1A). In CR2, he nine-gene ope on cu A is also loca ed,
which is unde he con ol o Co SR and includes he genes encoding he
MCO CuoA, he P
1B
- ype ATPase CopA, Co SR, and a p o ein ha
esembles he subuni III o he Cbb3- ype cy och ome C oxidase
(MXAN_3421) (S´
anchez-Su il e al., 2013). T ansc ip omic da a con i m
ha genes egula ed by Co SR a e implica ed in he main enance
esponse because he exp ession o he cu A ope on is main ained o e
ime (Fig. 1A). The gene o a hi d MCO, cuoC, is also loca ed nea o
CR2 and shows up egula ion by coppe as p e iously epo ed
(S´
anchez-Su il e al., 2007; Mo aleda-Mu˜
noz e al., 2010a) (Fig. 1A and
B).
Ou analyses also co obo a e he s ong up egula ion o he genes
coding o he e lux sys ems Cus2 and Cus3, and a mino induc ion o
cus1 and czc2 genes (Fig. 1A). Cus1 was epo ed o be mainly in ol ed
in Zn
2+
homeos asis (Mo aleda-Mu˜
noz e al., 2010a); he e o e, coppe
induc ion o he cus1 clus e is likely o be unspeci ic. To u he explo e
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
3
he ole o he Czc2 e lux pump in coppe homeos asis, we ob ained a
Δczc2 in- ame mu an using he M. xan hus DK1622 WT s ain as he
gene ic backg ound. To analyze he su i al o coppe oxici y, we
moni o ed he g ow h o he Δczc2 s ain in liquid CTT medium sup-
plemen ed wi h inc easing coppe concen a ions. The esul s showed
ha he Δczc2 mu an exhibi ed he same su i al pheno ype as he WT
s ain in bo h coppe -adap ed and non-adap ed cells (Fig. S2A). Gi en
ha high coppe concen a ions a ec mo ili y in M. xan hus
(G´
omez-San os e al., 2012), A- and S-mo ili y we e also e alua ed.
While A-mo ili y was una ec ed in he Δczc2 s ain (Fig. S2B), his
mu an displayed dec eased S-mo ili y when exposed o high coppe
concen a ions (Fig. S2C). Fu he mo e, Δczc2 s ain shows a sligh
Fig. 1. Genes coding p o eins in ol ed in M. xan hus coppe homeos asis. A: Hea map o he up egula ed DEGs in M. xan hus monocul u es and in co-cul u es wi h
S. melilo i a imes 2 h, 6 h and 10 h. Shaded ed g adien ep esen s Log2FC in gene exp ession. B: Func ion and sub-cellula loca ion o he up egula ed p o eins
implica ed in coppe de oxi ica ion. OM: ou e memb ane; IM: inne memb ane; HME_RND e lux pumps: Cus1, Cus2, Cus3 and Czc2; mul icoppe oxidases: CuoA,
CuoB and CuoC; P
1B
- ype ATPases: CopA and CopB; ou e memb ane p o ein: OmpB. The TCS Co SR egula es CopA and CuoA (in g een). The ECF sigma ac o Co E
con ols CopB, CuoB, and OmpB (in blue).
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
4
de ec in p eda ion ha becomes mo e appa en in he p esence o
coppe (Fig. S2D and E). This sugges s ha , al hough no essen ial o
coppe su i al, he Czc2 sys em con ibu es o he i ness o M. xan hus
in he p esence o his me al, po en ially alle ia ing he nega i e e ec s
o coppe on physiological p ocesses.
3.1.2. Down egula ed genes un a eled a new sys em wi hin CR1 likely
in ol ed in he acquisi ion o coppe and wo TCSs in ol ed in edox
homeos asis
Among he down egula ed genes in he p esence o coppe we e
hose o he clus e MXAN_0976-MXAN_0979 loca ed in CR1 (Figs. 2A
and C, S1A and B). Bioin o ma ic analyses o he genes in his clus e
e ealed ha MXAN_0976 and MXAN_0977 esemble mbnP and mbnH,
espec i ely. These wo-pa ne genes ypically co-occu in bac e ia and
a e e e ed o as he “me allo-mys e y pai ” (Manesis e al., 2021).
MbnP belongs o a coppe -binding p o eins amily, while MbnH is a
membe o he bac e ial diheme cy och ome c pe oxidases ha has been
shown o be loca ed in he pe iplasm o M. xan hus (Bha e al., 2011;
Manesis e al., 2021). The MbnPH pai has been p oposed o play a ole
in coppe edis ibu ion and acquisi ion, especially in me hano ophs
whe e i is in ol ed in coppe elease om he chalkopho e me h-
anobac in. Genes encoding o MbnPH pai s a e commonly associa ed
wi h genes encoding coppe -binding pe iplasmic chape ones and
memb ane anspo e s (Manesis e al., 2021). In ac , wo o he
down egula ed genes in M. xan hus a e hose coding o he P
1B
- ype
ATPase CopC (MXAN_0979) and a chlo ide channel p o ein
(MXAN_0978) (Fig. 2A). We p e iously ob ained h ee di e en ans-
la ional lacZ usions in he copC p omo e and none o hem exhibi ed
exp ession upon he addi ion o coppe . Mo eo e , a ΔcopC s ain did
no show any pheno ype in he p esence o coppe . We specula ed a ha
ime ha CopC could be in ol ed in coppe up ake, bu no exp ession
was de ec ed ei he a e he addi ion o me al chela o s
(Mo aleda-Mu˜
noz e al., 2010b). The cu en ansc ip omic da a ha e
shown ha , in ac , coppe down egula es he exp ession o he ans-
po e copC a 6 and 10 h (Fig. 2A). These esul s sugges an unknown
mechanism o coppe acquisi ion in M. xan hus in which MbnPH p o-
eins would hypo he ically ac o seques e coppe oge he wi h he
anspo e s copC and/o MXAN_0978 (Fig. 2A and C; Table S4). To
u he in es iga e, a ΔcopC in- ame dele ion mu an was ob ained om
he DK1622 s ain and i s pheno ype was analyzed unde
coppe -de icien condi ions. A e 48 h o incuba ion in he p esence o
e a hiomolybda e (TTM), a speci ic Cu²⁺ chela o , he ΔcopC s ain was
sligh ly mo e sensi i e han he WT s ain o coppe chela ion (Fig. 2D).
This would indica e ha while copA and copB a e in ol ed in coppe
e lux, copC migh be hypo he ically in ol ed in he esponse o coppe
s a a ion. Al hough he e a e some epo s on ATPase-ca alyzed coppe
up ake (Hassani e al., 2010), coppe acquisi ion by P
1B
- ype ATPases
has ne e been di ec ly demons a ed and equi es u he in es iga ion.
Al e na i ely, CopC migh be p ocu ing coppe o MnbPH in he pe i-
plasm, so hey can ealloca e he me al unde coppe s a a ion
Fig. 2. Down egula ion o M. xan hus genes in he p esence o coppe . Hea map o down egula ed DEGs in he copC clus e (A) and genes encoding wo pa alogous
egBA-like TCS (B) in M. xan hus monocul u es and in co-cul u es wi h S. melilo i a imes 2 h, 6 h and 10 h. Shaded blue g adien ep esen s Log2FC in gene
exp ession. C: P edic ed unc ion and sub-cellula loca ion o p o eins encoded by copC clus e genes ha seem o be implica ed in coppe acquisi ion in he absence
o his me al. P o eins ha e been colo ed as in Fig. S1 o be e loca e hem in hei genomic en i onmen s. D: G ow h o WT and ΔcopC s ains a e 48 h in CTT
media supplemen ed wi h di e en concen a ions o he Cu
2+
chela o TTM. Da a ep esen he a e age ±s anda d de ia ion o h ee eplica es.
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
5
(Fig. 2D).
Besides he genes in he CR1, wo addi ional gene clus e s
(MXAN_6223-MXAN_6227 and MXAN_6976-MXAN-6981), bo h
including genes ha esemble he RegBA TCS om o he bac e ia, s and
ou among he mos down egula ed genes in he p esence o coppe
(Fig. 2B; Fig. S1A and B). These TCSs a e highly conse ed global eg-
ula o y sys ems ha con ol he edox s a e in a a ie y o ene gy-
gene a ing and ene gy-u ilizing biological p ocesses, such as elec on
anspo and ae o axis (Wu and Baue , 2008). A se o genes coding o
oxido educ ases and cy och ome c biogenesis p o eins we e also
down egula ed in he p esence o coppe (Table S4) and up egula ed
du ing p eda ion, when bo h egBA clus e s we e also up egula ed
(P´
e ez e al., 2022). Thus, i is emp ing o specula e ha hese TCSs a e
egula ing p o eins implica ed in elec on anspo unde changed
coppe le els.
3.1.3. Oxida i e s ess esponse and coppe oxici y
When coppe en e s he bac e ial cell, he educing en i onmen in
he cy oplasm changes Cu
2+
o Cu
+
, which can hen pa icipa e in
Fen on- ype eac ions and he Habe -Weiss cycle o p oduce highly
eac i e oxygen species (ROS) ha , in u n, eac wi h lipids, p o eins,
and nucleic acids. Coppe -induced oxida i e s ess can also lead o hiol
oxida ion, causing he inac i a ion o hiol-con aining p o eins and he
deple ion o glu a hione (GSH), which plays a majo p o ec i e ole
agains hea y me al oxici y. Howe e , one o he main coppe - oxici y
mechanisms is he displacemen o i on om i on-sul u clus e s and
p o ein misme alla ion, esul ing in hei inac i a ion (And ei e al.,
2020; Li e al., 2021).
Ou ansc ip omic da a indica e ha p o eins con aining hio-
edoxin domains, which a e in ol ed in he educ ion o a numbe o
enzymes main aining hiol-based edox homeos asis (Tani uji and
Kimu a, 2024), a e he main de ensi e esponse agains coppe -induced
oxida i e s ess. O he M. xan hus genes encoding p o eins wi h hio-
edoxin domains, MXAN_6156 and MXAN_7304 we e s ongly up egu-
la ed, while MXAN_0289, MXAN_0290, MXAN_2652 and MXAN_6848
we e sligh ly induced (Fig. 3A and C; Table S4). To u he alle ia e hiol
s ess, in he p esence o coppe , M. xan hus also induces he genes
coding o a GSH disul ide educ ase (MXAN_2318) and a GSH S- ans-
e ase (MXAN_5471) (Fig. 3A and C), p o eins ha a e commonly
in ol ed in he de oxi ica ion o oxidized GSH (Lu and Holmg en, 2014).
The GSH esponse du ing coppe -induced oxida i e s ess seems o be
limi ed o main aining he edox balance o he p eexis ing GSH a he
han inc easing he syn hesis o new GSH (Okada and Kimu a, 2022),
since we did no obse e signi ican changes in he exp ession o he
Fig. 3. M. xan hus esponse o coppe -induced oxida i e s ess. Up egula ed DEGs in ol ed in an ioxidan sys ems, including genes o he hio edoxin sys em (T x)
and he glu a edoxin an ioxidan sys em (Go ) (A), and ROS-sca enging enzymes (B) ha con ibu e o edox homeos asis. C: Hea map o DEGs in ol ed in oxida i e
s ess caused by coppe in M. xan hus monocul u es and in co-cul u es wi h S. melilo i a imes 2 h, 6 h and 10 h. Shaded ed o blue g adien s ep esen Log2FC
up egula ion o down egula ion, espec i ely, in gene exp ession.
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
6
main genes o GSH syn hesis, such as he glu ama e-cys eine ligase genes
gcl1 and gcl2 (MXAN_1276 and MXAN_5806), no he glu ha hione
syn he ase MXAN_0017.
In he main enance o edox homeos asis, we also obse ed he
up egula ion o M. xan hus genes encoding o ROS-sca enging enzymes,
such as he ca alase Ka E (MXAN_6188) (Fig. 3B and C). Al hough
coppe Fen on-like eac ions do no p oduce H
2
O
2
, he de oxi ica ion o
supe oxide ions by supe oxide dismu ases (such as hose coded by he
up egula ed genes sodB and sodC) esul s in he p oduc ion o H
2
O
2
. The
gene clus e MXAN_1563–1564, which encodes o wo alkyl hyd o-
pe oxide educ ases (AhpD and AhpC, espec i ely), is also up egula ed
unde coppe -induced s ess, and suppo ca alases in educing H
2
O
2
and o ganic hyd ope oxides (Thomas e al., 2008) (Fig. 3B and C).
As men ioned abo e, one o he main mechanisms o coppe oxici y
is he displacemen o i on ions om he i on-sul u clus e s. This is
especially he case o dehyd a ase enzymes ha a e pa o many
me abolic pa hways (Macombe and Imlay, 2009). In ac , in he p es-
ence o coppe , se e al genes encoding o dehyd a ases implica ed in
he p oduc ion o M. xan hus seconda y me aboli es, such as myxo i -
escin (MXAN_3931–3950), myxoch omide (MXAN_4077–4079),
DKxan hene (MXAN_4293–4304) o myxalamide (MXAN_4525–4530),
we e down egula ed (Table S4), sugges ing ha coppe inhibi s he
syn hesis o hese p oduc s, al hough how coppe a ec s hese gene
clus e s a he ansc ip ional le el is s ill unknown.
Sul u me abolism accomplishes essen ial cell unc ions, and i has
been equen ly epo ed o be in ol ed in coppe esis ance and in he
main enance o edox homeos asis in coope a ion wi h o he de oxi i-
ca ion sys ems (Huang e al., 2019; Yan e al., 2021). The
hio edoxin-dependen adenylylsul a e educ ase MXAN_2340, in ol ed
in assimila o y sul a e educ ion o sul ide, as well as he gene clus e
MXAN_0177–0179, encoding a sul i e expo e , a hodanese-like
domain-con aining p o ein, and a me allo-hyd olase, espec i ely,
we e down egula ed in M. xan hus in he p esence o coppe (Table S4).
On he o he hand, he clus e MXAN_4309–4316, including genes
encoding a sul a e expo e and a Ds E amily p o ein in ol ed in sul u
oxida ion, was up egula ed in he p esence o coppe (Table S4).
3.2. M. xan hus coppe esponse du ing p eda ion
P e iously, we demons a ed ha coppe plays a ole du ing he
in e ac ion be ween M. xan hus and S. melilo i (Con e as-Mo eno e al.,
2020). He e, we in es iga ed how his me al in luences his in e ac ion
by compa ing myxobac e ial DEGs in co-cul u es a di e en ime-poin s
in he p esence o coppe wi h da a ob ained om he M. xan hus
monocul u es.
As in monocul u es, he highes induced genes we e hose di ec ly
implica ed in coppe e lux and de oxi ica ion, in ol ing genes in CR1
and CR2, as well as he genes o he HME-RND sys ems Czc2 and Cus3,
and he e i in genes MXAN_6156 and MXAN_7304 (Figs. 1A, 3C, and
S1; Table S4). When compa ed o he FPKM alues o monocul u es, he
da a e eal ha he exp ession o mos genes is no signi ican ly al e ed
by he p esence o p ey. Howe e , he h ee genes coding o he Cus3
e lux sys em had lowe FPKM alues du ing p eda ion in he p esence o
he me al (Fig. S3A; Table S3). Since p eda ion has been shown o a ec
he spa ial dis ibu ion o coppe in M. xan hus colonies and he oxida-
i e s ess esponse (Con e as-Mo eno e al., 2018), i is possible ha
he ansc ip ional mechanisms behind cus3 coppe induc ion a e
a ec ed by hese condi ions.
On he o he hand, he exp ession o se e al genes seems o be
al e ed by coppe only unde p eda ion condi ions (Table S4). To disce n
whe he hese changes a e caused by a me e al e a ion o he p eda o y
exp ession pa e n by he p esence o coppe , o i hey a e pa o a mo e
speci ic syne gis ic esponse, we compa ed he di e en ial exp ession o
hese genes in M. xan hus p eda osomes du ing p eda ion on S. melilo i in
he absence o coppe (Fig. S4). One o he mos in e es ing obse a ions
unde hese condi ions was he up egula ion a e 2 h o he gene clus e
MXAN_5264-MXAN_5265 coding o a p o ein wi h a ca ion e lux
domain and a p o ein wi h a glyoxal oxidase domain, espec i ely
(Fig. S4A; Table S4). This gene clus e is in ol ed in me al handling and
is egula ed by he Co E-pa alog Co E2 (MXAN_5263), which was p e-
iously ound o be ac i a ed by zinc and cadmium a he han coppe
(Ma cos-To es e al., 2016). As s a ed abo e, me als such as coppe o
i on play an impo an ole du ing p eda ion, and bo h pa ne s in his
in e ac ion can ei he compe e o acqui e hem o de end hemsel es
om me al oxici y, which is bound o al e in acellula me al luc u-
a ions. This would explain why coppe , ei he di ec ly o indi ec ly, is
able o ac i a e Co E2 unde hese condi ions. Whe he
coppe -dependen exp ession o he Co E2 egulon exclusi ely du ing
p eda ion is a consequence o an in acellula me al imbalance, o i
hese genes a e equi ed o be exp essed unde hese condi ions, hin ing
a a mo e speci ic ole du ing p eda ion is s ill o be de e mined.
A e six hou s, a gene clus e coding o a glycine be aine/L-p oline
ABC anspo sys em (MXAN_2248–2251) was also speci ically up e-
gula ed by coppe du ing p eda ion (Figs. S3B and S4A; Table S3 and
S4). Bo h be aine and p oline a e osmo ole an molecules (T ipa hi
e al., 2022), and he up egula ion o his anspo e sugges s a ole in
main aining osmo ic balance du ing p eda ion, which seems o be o
mo e ele ance when in e ac ion occu s in coppe - ich en i onmen s.
The anspo e ’s ole in main aining osmo ic balance could be i al no
only o su i al bu also o op imizing he p eda o y beha io o
M. xan hus in hese ha sh condi ions.
Among he ansc ip s induced by coppe exclusi ely du ing p eda-
ion s and ou wo glycosyl ans e ases (MXAN_1422 and MXAN_2921),
p obably in ol ed in modi ying cell su ace componen s, such as poly-
saccha ides o lipopolysaccha ides, which could play a ole in main-
aining s uc u al in eg i y du ing p eda ion o acili a ing in e ac ions
wi h he p ey. The selec i e induc ion o wo me allop o eins
(MXAN_2692 and MXAN_6714) in co-cul u es wi h coppe , which could
play oles in me al binding, de oxi ica ion, o ca aly ic unc ions unde
me al s ess, ein o ces he idea ha ce ain me allop o eins a e essen-
ial o managing coppe du ing p eda ion. A clus e o genes in ol ed in
lipid biosyn hesis (MXAN_6398–6404) is down egula ed in mono-
cul u es bu up egula ed by coppe du ing p eda ion, e lec ing how
eac i e lipid composi ion is o changes in en i onmen al condi ions.
This esul aligns wi h p e ious indings by P´
e ez e al. (2022) and Jain
e al. (2025), who epo ed simila me abolic shi s in esponse o
p eda o y in e ac ions. This up egula ion o lipid biosyn hesis genes
only du ing p eda ion could be indica i e o se e al adap i e esponses,
such as inc eased memb ane emodeling, ene gy s o age, o signaling
pa hways. On he o he hand, wo ansc ip ional egula o s
(MXAN_6233 and MXAN_6479) and a clus e ha includes he hyb id
his idine kinase (MXAN_0095) and a me hyl ans e ase
domain-con aining p o ein (MXAN_0096) we e only induced in he
p esence o bo h coppe and p ey, and could be pa o he signaling
pa hways in ol ed in modula ing s ess esponse, me abolism adjus -
men s, o in e ac ion wi h S. melilo i unde coppe condi ions (Fig. S4A;
Table S4).
Rega ding he ansc ip s ha a e down egula ed by coppe du ing
p eda ion, i is in e es ing o no e he down egula ion o he ca alase
gene ka E (MXAN_6188), which, as indica ed abo e, is up egula ed by
coppe in monocul u es. Ka E is he main ca alase du ing bo h expo-
nen ial g ow h and he s a iona y phase, as well as du ing ui ing body
o ma ion, and appea s o be essen ial o M. xan hus iabili y (Kimu a
e al., 2022). The down egula ion o he ka E gene, along wi h ha o
many oxida i e s ess esponse genes in he con ex o p eda ion and
coppe (Fig. 3C), sugges s lowe in acellula le els o H
2
O
2
, indica ing
ha he combined esponse o bo h condi ions enhances he oxida i e
s ess i ness o M. xan hus du ing p eda o -p ey dynamics. The only
up egula ed gene ela ed o he oxida i e s ess esponse in his con ex
is MXAN_4389 (Fig. S4A), which codes o he ca alase Ka B, which has
been epo ed o be apidly induced by H
2
O
2
(Kimu a e al., 2022). The
induc ion o his ca alase a a la e s age o p eda ion in he p esence o
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
7
coppe sugges s ha i may play a p o ec i e ole by acili a ing he as
deg ada ion o ex e nal H
2
O
2
pe mea ing h ough he cell memb ane, as
sugges ed by Kimu a e al. (2022). We p e iously epo ed ha S. melilo i
esponds ac i ely o p eda ion by up egula ing H
2
O
2
p oducing genes
(So o e al., 2023), and i is possible ha he p esence o coppe could
u he enhance he p oduc ion o his ROS in he p ey, which in u n
may ac i a e ka B.
Simila ly, he clus e MXAN_2948-MXAN_2951, encoding o a me al
ABC anspo e sys em, is down egula ed by coppe only while p eying
on S. melilo i. This, oge he wi h he abo emen ioned lowe exp ession
le els o he Cus3 sys em and he ca alase Ka E, could sugges ha
ac i a ion o he coppe esponse du ing p eda ion may imp o e he
p eda o physiological condi ions, esul ing in lowe in e nal oxida i e
s ess and a mo e e icien me al handling han p eda ing in he absence
o coppe .
3.3. Coppe in e e ence wi h i on up ake and a icking
The exp ession p o ile o he i on up ake machine y in M. xan hus
unde coppe s ess, in bo h monocul u es and co-cul u es, e eals
in iguing dynamics ha highligh he in ica e ela ionship be ween
coppe and i on me abolism. Mos o he genes in ol ed in i on
compe i ion du ing myxobac e ial p eda ion (Con e as-Mo eno e al.,
2024b) show a dis inc pa e n o down egula ion in monocul u es a 2
and 6 h, ollowed by an inc ease in exp ession a 10 h (Fig. S5), which
sugges s ha he p esence o coppe signi ican ly impac s i on homeo-
s asis. This obse a ion can be explained by se e al mechanisms based
on he syne gis ic i on-coppe oxici y: i) C oss- alk be ween
i on- egula o y p o eins and coppe . The majo i on homeos asis egu-
la o Fu A in M. xan hus esponds o i on a ailabili y and con ols he
exp ession o i on-up ake genes (Con e as-Mo eno e al., 2024b).
Howe e , high coppe le els can cause he misme alla ion o Fu A
and/o o he i on-sensing p o eins, dis up ing hei unc ion and leading
o he imp ope egula ion o i on homeos asis genes. ii) Coppe and i on
a e known o ca alyze he o ma ion o ROS, causing signi ican oxida-
i e s ess. In esponse o he excess o coppe , M. xan hus mus igh ly
egula e he le els o bo h me als o a oid oxida i e damage. Addi-
ionally, coppe -induced ROS (e.g., supe oxide and hyd ogen pe oxide)
can also dis up he p ope me alla ion o i on-dependen enzymes and
ansc ip ional egula o s, u he complica ing i on me abolism (Imlay,
2014). The empo a y down egula ion o i on up ake a ea lie s ages (2
and 6 h) may se e as a p o ec i e mechanism agains i on-coppe
syne gis ic oxici y. iii) Coppe and side opho es linkage. M. xan hus
p oduces myxochelin, a ca echola e- ype side opho e ha pa icipa es
in i on acquisi ion. In e es ingly, some side opho es, such as ye sinia-
bac in om Esche ichia coli and en e obac in om Salmonella en e ica,
can also bind Cu
2+
, enhancing coppe esis ance and p e en ing i s
educ ion o he mo e oxic Cu
+
o m (Hy e e al., 2021).
Simila o o he bac e ia, such as Pseudomonas ae uginosa, which
inc eases i s equi emen o i on o su i e unde coppe s ess
(Chillappaga i e al., 2010), M. xan hus may also ace a highe demand
o i on du ing coppe exposu e. Mo eo e , he p esence o coppe
du ing p eda ion on S. melilo i u he al e s he exp ession pa e n o
i on up ake genes, shi ing owa d an ea lie up egula ion o hese
mechanisms, deno ing how c i ical i on compe i ion becomes o
M. xan hus du ing p eda ion (Fig. S5). This ensu es ha M. xan hus can
no only wi hs and coppe s ess bu also ou compe e S. melilo i o
limi ed i on esou ces (Con e as-Mo eno e al., 2024b). In ac , he
compe i ion o i on in ensi ied as p eda ion p og essed, equi ing an
inc ease in i on up ake a la e ime poin s (Fig. S5). Mo eo e , i has
been ecen ly demons a ed ha high i on concen a ions p e en
cellula coppe accumula ion and oxici y in Caulobac e , whe e some
p o eins in ol ed in i on anspo p o ide p o ec ion agains coppe
excess (Che y e al., 2025).
3.4. Coppe and an ibio ic co-selec ion
Since soil en i onmen s can each high coppe concen a ions om
he use o his me al in ag icul u e as a biocide and om na u al sou ces
(up o 496 and 1817 mg Cu kg
−1
has been epo ed in he opsoil o some
Eu opean [Ballabio e al., 2018] and sou he n B azilian [Ko chagin
e al., 2020] ag icul u al si es, espec i ely), we wan ed o in es iga e
how he coppe esponse in M. xan hus could con ibu e o he selec ion
o ARGs ha could sp ead among soil popula ions. Fo his pu pose, he
M. xan hus WT s ain was g own in liquid medium and he suscep ibili y
o he cells o a panel o 11 an ibio ics (Table S2) wi h di e en mech-
anisms o ac ion was analyzed in media supplemen ed wi h 300
μ
M
CuSO
4
. The esul s ob ained indica e ha , in he p esence o coppe ,
M. xan hus WT cells inc eased hei suscep ibili y o kanamycin, and also
sligh ly o e acycline and nalidixic acid (Fig. 4A). These esul s imply
ha coppe , e en below oxic le els, enhances he e ec i eness o hese
an ibio ics agains M. xan hus, sugges ing a syne gis ic e ec o coppe
wi h an ibio ics as biocides. This, howe e , can also p omo e he
exp ession and selec ion o ARGs a lowe an ibio ic concen a ions o
su i e unde hese condi ions. As discussed abo e, he coppe esponse
is a mul i ac o ial p ocess in ol ing he di e en ial exp ession o hun-
d eds o genes and, while some o hose genes may inc ease an ibio ic
sensi i i y, o he genes may con ibu e o inc ease esis ance and hus be
suscep ible o sp eading an ibio ic esis ance among soil popula ions.
Since he iden i ied genes in ol ed in he coppe esponse o
M. xan hus a e no loca ed in mobile gene ic elemen s, coppe and
an ibio ic esis ance co-selec ion can only be media ed by c oss-
esis ance and co- egula ion e en s in his bac e ium. To app oach he
co-selec ion o coppe and an ibio ic esis ance genes, we s udied he
con ibu ion o an ibio ic esis ance o he 7 e lux sys ems ( o c oss-
esis ance) and 2 ansc ip ional egula o s ( o co- egula ion) ha
ha e been shown o be induced by coppe in M. xan hus. Fo his pu -
pose, we u ilized dele ion mu an s o he genes encoding he P
1B
- ype
ATPases CopA, CopB, and CopC, he e lux sys ems Cus1, Cus2, Cus3 and
Czc2, and he ansc ip ional egula o s Co SR and Co E (Table S1), and
analyzed hei esis ance in he p esence o 300 µM CuSO
4
agains he
di e en an ibio ics used p e iously wi h he M. xan hus WT s ain
(Table S2).
The analysis o he an ibio ic suscep ibili y o M. xan hus mu an
s ains o he coppe - esponsi e egula o s Co SR and Co E e ealed
small di e ences om he WT s ain (Fig. S6A). Thus, he Δco SR s ain
was mo e sensi i e o kanamycin han he WT s ain. Simila ly, a mu an
in he ATPase gene copA, egula ed by Co SR, also displayed a sligh
inc ease in sensi i i y o kanamycin compa ed wi h he WT s ain
(Fig. S6B), sugges ing ha Co SR a ec s esis ance o his an ibio ic ia
copA induc ion. In a simila manne , he Δco E s ain and he mu an o
he Co E- egula ed ATPase gene copB we e mo e sensi i e o e y h o-
mycin (Fig. S6A and B). The Δco E mu an was also sligh ly mo e sen-
si i e o moxi loxacin (Fig. S6A), which would poin o an addi ional
gene in he Co E egulon in ol ed in he esis ance o his an ibio ic. As
o he hi d ATPase gene, copC, he mu an showed no no able changes
in an ibio ic sensi i i y, indica ing ha his e lux p o ein is no in ol ed
in coppe -an ibio ic c oss- esis ance (Fig. S6B).
On he o he hand, he ou e lux pumps in ol ed in coppe ho-
meos asis showed a mo e signi ican ole in an ibio ic esis ance. Among
hem, he Δczc2 mu an only displayed a low inc ease in esis ance o
nalidixic acid, moxi loxacin, and e y h omycin (Fig. 4D). As discussed
abo e, his e lux pump plays a mino ole in coppe esis ance, mainly
a ec ing social mo ili y in he p esence o coppe . This sligh inc ease in
he an ibio ic esis ance o he Δczc2 mu an may be due o he up e-
gula ion o o he coppe - esis ance mechanisms ha may con e esis-
ance o hese an ibio ics. The Cus1 and Cus2 e lux pumps exhibi ed a
s iking coppe -an ibio ic c oss- esis ance, as Δcus1 and Δcus2 s ains
a e mo e suscep ible o e acycline an ibio ics (doxycycline, e acy-
cline, and oxy e acycline) compa ed wi h he WT s ain (Fig. 4B and C).
These esul s a e consis en wi h p e ious epo s o coppe and
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
8
e acycline c oss- esis ance, and wi h e lux being one o he mos
common mechanisms o e acycline esis ance, wi h RND anspo e s
commonly media ing he in insic e lux o e acyclines (G ossman,
2016). Addi ionally, he Δcus2 s ain showed inc eased sensi i i y o
kanamycin as well, highligh ing he po en ial in ol emen in b oade
an ibio ic esis ance mechanisms o he Cus2 e lux sys em. I should be
no ed ha he assays wi h he Δcus2 s ain we e pe o med in media
supplemen ed wi h a lowe coppe concen a ion (50 µM CuSO
4
)
because o he highe sensi i i y o his mu an o he p esence o he
me al (Mo aleda-Mu˜
noz e al., 2010a).
An unexpec ed inding in ol ed he highly coppe -induced Cus3
pump. Con a y o he Δcus2 s ain, al hough he Δcus3 s ain is sensi-
i e o high coppe concen a ions (Mo aleda-Mu˜
noz e al., 2010a), i
does no display al e ed suscep ibili y o he coppe concen a ions
assayed he e. Su p isingly, his mu an was ully esis an o nalidixic
acid unde hese condi ions (Fig. 4B). To quan i iy his s iking esul ,
MIC o nalidixic acid was de e mined o compa e he le el o esis ance
in he WT and Δcus3 s ains in he p esence o coppe , ob aining MIC
alues o 50 and 800 µg/ml, espec i ely. Since in he absence o coppe ,
when cus3 is no induced, M. xan hus shows suscep ibili y o nalidixic
acid (Fig. 4A), his esul sugges s ha Cus3 dis up ion leads o changes
ha inad e en ly p o ide esis ance o nalidixic acid. Al hough o a
lesse ex en , ou esul s also show ha he Δcus3 s ain is also mo e
esis an o e y h omycin, sugges ing ha highe an ibio ic esis ance is
ela ed o he physiological s a e o he s ain a he han speci ically
coun e ac ing he mode o ac ion o hese an ibio ics. This mechanism
has been obse ed in o he bac e ial species, whe e dis up ions in
ce ain cellula sys ems can enhance esis ance o an ibio ics by al e ing
memb ane pe meabili y o ene gy dynamics. In p e ious s udies, simila
dis up ions in memb ane-associa ed sys ems led o an ibio ic esis ance
h ough indi ec pa hways (Wales and Da ies, 2015). This may explain
he unexpec ed esis ance o he Δcus3 mu an , highligh ing a possible
compensa o y mechanism wi hin M. xan hus cells. In ac , we p e iously
demons a ed ha he dele ion o some coppe homeos asis genes, such
as hose o he CopA o CopB ATPases, induces a compensa o y
mechanism ha al e s he exp ession p o iles o o he genes, such as
hose o he Cus2 and Cus3 sys ems (Mo aleda-Mu˜
noz e al., 2010b).
In summa y, his analysis highligh s he complex in e play be ween
coppe homeos asis and an ibio ic esis ance in M. xan hus, wi h he
me al-e lux pumps Cus1 and Cus2 being he main playe s in luencing
c oss- esis ance o an ibio ics. Fu he esea ch is needed o elucida e
he p ecise mechanisms by which hese Cus- ype pumps con ibu e o
coppe -an ibio ic in e ac ions.
4. Conclusions
The complex M. xan hus coppe esponse includes an unusual num-
be o inducible elemen s in ol ed in e lux ( wo ATPases and ou RND-
ype sys ems), coppe oxida ion ( h ee MCOs), and se e al coppe -
binding p o eins and ou e memb ane p o eins. T ansc ip omic da a
allowed he iden i ica ion o a clus e , ha includes he gene o he
ATPase CopC, which may be in ol ed in coppe edis ibu ion and/o
up ake unde coppe - es ic ed condi ions. Addi ionally, he da a un-
eiled a mul i ace ed esponse o oxida i e s ess, elici ed mainly
h ough he ac i a ion o se e al hio edoxins and in e e ences in i on
me abolism.
Fig. 4. Coppe -an ibio ic c oss- esis ance in M. xan hus s ains. A: An ibio ic sensi i i y o he WT s ain in he absence o p esence o 300 µM CuSO
4
. B: An ibio ic
sensi i i y o he WT, Δcus1 and Δcus3 s ains in media supplemen ed wi h 300 µM CuSO
4
. C: An ibio ic sensi i i y o he WT and Δcus2 s ains g owing in he
p esence o 50 µM CuSO
4
. D: An ibio ic sensi i i y o he WT and Δczc2 s ains in he p esence o 300 µM CuSO
4
. An ibio ic es ed: NA, nalidixic acid; CIP,
cip o loxacine; LEV, le o loxacin; MXF, moxi loxacin; DO, doxycycline; TE, e acycline; OT, oxy e acycline; K, kanamycin; E, e y h omycin; AZM, azi h omycin; SP,
spi amycin. See Table S2 o he concen a ions assayed. Th ee independen eplica es we e analyzed, and da a ep esen a e age ±s anda d de ia ions. The wo-
ailed S uden ’s - es was used o de e mine signi ican di e ences (*: P<0.05;
**
: P<0.01;
***
: P<0.005;
****
: P<0.001).
F.J. Ma cos-To es e al.
Mic obiological Resea ch 302 (2026) 128357
9
