micb-11-00094 Feb ua y 1, 2020 Time: 12:20 # 1
ORIGINAL RESEARCH
published: 04 Feb ua y 2020
doi: 10.3389/ micb.2020.00094
Edi ed by:
Ha i S. Mis a,
Bhabha A omic Resea ch Cen e
(BARC), India
Re iewed by:
Alessio Mengoni,
Uni e si y o Flo ence, I aly
Sunil D. Sa oj,
Symbiosis In e na ional Uni e si y,
India
*Co espondence:
Au elio Mo aleda-Muñoz
au eliom@ug .es
Special y sec ion:
This a icle was submi ed o
Mic obial Physiology and Me abolism,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 15 No embe 2019
Accep ed: 16 Janua y 2020
Published: 04 Feb ua y 2020
Ci a ion:
Con e as-Mo eno FJ,
Muñoz-Do ado J, Ga cía-Tomsig NI,
Ma ínez-Na ajas G, Pé ez J and
Mo aleda-Muñoz A (2020) Coppe
and Melanin Play a Role
in Myxococcus xan hus P eda ion on
Sino hizobium melilo i.
F on . Mic obiol. 11:94.
doi: 10.3389/ micb.2020.00094
Coppe and Melanin Play a Role in
Myxococcus xan hus P eda ion on
Sino hizobium melilo i
F ancisco Ja ie Con e as-Mo eno1, José Muñoz-Do ado1,
Na alia Isabel Ga cía-Tomsig1,2, Gonzalo Ma ínez-Na ajas1, Juana Pé ez1and
Au elio Mo aleda-Muñoz1*
1Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada, G anada, Spain, 2Es ación Expe imen al
del Zaidín, G anada, Spain
Myxococcus xan hus is a soil myxobac e ium ha exhibi s a complex li ecycle wi h
wo mul icellula s ages: coope a i e p eda ion and de elopmen . Du ing p eda ion,
myxobac e ial cells p oduce a wide a ie y o seconda y me aboli es and hyd oly ic
enzymes o kill and consume he p ey. I is known ha euka yo ic p eda o s, such as
ameba and mac ophages, in oduce coppe and o he me als in o he phagosomes o
kill hei p ey by oxida i e s ess. Howe e , he ole o me als in bac e ial p eda ion has
no ye been es ablished. In his wo k, we ha e add essed he ole o coppe du ing
p eda ion o M. xan hus on Sino hizobium melilo i. The use o biosenso s, a iable
p essu e scanning elec on mic oscopy, high- esolu ion scanning ansmission elec on
mic oscopy, and ene gy dispe si e X ay analysis has e ealed ha coppe accumula es
in he egion whe e p eda o and p ey collide. This accumula ion o me al up- egula es
he exp ession o se e al mechanisms in ol ed in coppe de oxi ica ion in he p eda o
( he P1B-ATPase CopA, he mul icoppe oxidase CuoA and he ipa i e pump Cus2),
and he p oduc ion by he p ey o coppe -inducible melanin, which is a polyme wi h he
abili y o p o ec cells om oxida i e s ess. We ha e iden i ied wo genes in S. melilo i
(encoding a y osinase and a mul icoppe oxidase) ha pa icipa e in he biosyn hesis
o melanin. Analysis o p ey su i abili y in he co-cul u e o M. xan hus and a mu an o
S. melilo i in which he wo genes in ol ed in melanin biosyn hesis ha e been dele ed
has e ealed ha his mu an is mo e sensi i e o p eda ion han he wild- ype s ain.
These esul s indica e ha coppe plays a ole in bac e ial p eda ion and ha melanin
is used by he p ey o de end i sel om he p eda o . Taking in o conside a ion ha
S. melilo i is a ni ogen- ixing bac e ium in symbiosis wi h legumes ha coexis s in
soils wi h M. xan hus and ha coppe is a common me al ound in his habi a as a
consequence o se e al human ac i i ies, hese esul s p o ide clea e idence ha he
accumula ion o his me al in he soil may in luence he mic obial ecosys ems by a ec ing
bac e ial p eda o y ac i i ies.
Keywo ds: Myxococcus xan hus, p eda ion, coppe , melanin, Sino hizobium melilo i
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
INTRODUCTION
Myxococcus xan hus is a soil bac e ium wi h a peculia
mul icellula beha io , which is obse ed in all s ages o i s
li ecycle. In he absence o nu ien s, cells o ganize gliding
mo emen s o build mac oscopic ui ing bodies, wi h h ee
subpopula ions o cells showing di ision o labo (Muñoz-
Do ado e al., 2016). In hei na u al habi a s, hey o m
communi ies named swa ms ha eed by p eying on a la ge
a ie y o mic oo ganisms, including G am-posi i e and G am-
nega i e bac e ia, and some euka yo ic mic oo ganisms (Pé ez
e al., 2016). M. xan hus swa ms mo e owa d hei p ey and once
hey a e nea by hey ca y ou a coope a i e p eda ion p ocess
whe e he bac e ial communi y sec e es hyd oly ic enzymes and
a la ge numbe o seconda y me aboli es ha immobilize, kill
and deg ade he p ey. Many o he p eda o y molecules a e
packaged wi hin ou e memb ane esicles, mos likely o acili a e
hei anspo and o educe he isk o exploi a ion by chea e s
(Whi wo h, 2011;Be leman e al., 2014;Remis e al., 2014).
The hyd olyzed p oduc s a e used by he p eda o o g ow
(Keane and Be leman, 2016;Muñoz-Do ado e al., 2016;Pé ez
e al., 2016). Consequen ly, bac e ial p eda ion is a mul i ace ed
p ocess, which depends on nume ous pa ame e s associa ed
wi h bo h he p eda o and p ey. Al hough all hese elemen s
in luence he p eda o y in e ac ion, mos likely none o hem is
essen ial o i .
Bac e ial p eda ion is inc easingly being pos ula ed as
a c ucial ac o in biodi e si y due o i s po en ial ole
in con olling and shaping bac e ial popula ions in he
en i onmen (Chase e al., 2002;Galle e al., 2007;Nai
e al., 2019). In bac e ial p eda o y p ocesses, i is no only
he p eda o ha plays an ac i e ole in a acking and killing
he p ey, he cha ac e is ics o he p ey a e also decisi e.
Du ing he p og ession o he in e ac ion, he p eda o also
p omp s a a ie y o p ey esponses and adap a ions ha help
o esis o escape p eda ion. M. xan hus e icien ly p eys on
se e al soil bac e ia o high ag icul u al and bio echnological
alue, such as S ep omyces,Sino hizobium, Esche ichia,
and Bacillus. In he case o S ep omyces coelicolo , cells
espond wi h an ibio ic o e p oduc ion and by al e ing
mul icellula de elopmen (Pé ez e al., 2011). The p esence
o galac oglucan p o ec s Sino hizobium melilo i om being
killed by M. xan hus (Pé ez e al., 2014), and co-e olu ion
expe imen s wi h M. xan hus and Esche ichia coli as i s p ey
ha e demons a ed ha he p eda o induces changes o
p ey ai s such as he p oduc ion o mucus and he ou e
memb ane p o ease OmpT (Nai e al., 2019). In he case
o Bacillus,B. sub ilis p oduces bacillaene and spo e- illed
megas uc u es in esponse o p eda ion (Mülle e al., 2014,
2015), while B. licheni o mis escapes om M. xan hus p eda ion
by deac i a ing he an ibio ic myxo i escin (Wang e al., 2019).
A ansc ip omic s udy using E. coli as he p ey epo ed ha
he p esence o he p eda o induced signi ican changes in
40% o he p ey genes. Mo eo e , i e me abolic pa hways
we e signi ican ly up- egula ed in E. coli upon exposu e o
ou e memb ane esicles, supe na an and/o p eda o y cells
(Li ings one e al., 2018).
These in e species in e ac ions a e also a ec ed by
en i onmen al changes o luc ua ing concen a ions o elemen s
such as me als. Coppe is a ansi ion me al ha accumula es
in soils due o an h opogenic ac i i ies, since i is widely
used in a ious indus ies (elec ic cables, mo o s, gene a o s,
a mamen s, wa e anspo , deco a ion, coins, e c.). On he
o he hand, he use o coppe is widesp ead due o i s b oad
spec a ac i i y agains bac e ia, i uses, yeas s and o he ungi.
I is added as a ood supplemen o s imula e animal g ow h
by in luencing he in es inal mic obio a. Coppe is used on
hospi al su aces since i p o ides p o ec ion agains in ec ious
mic obes. Cu en ly, coppe is also allowed in a bo icul u e,
i icul u e, ho icul u e, ex ensi e ag icul u e, and e en o ganic
ag icul u e, as a ungicide and he bicide. The Eu opean Food
Sa e y Au ho i y (EFSA) has published a epo ha wa ns o he
high isk posed by coppe o bi ds, mammals and soil o ganisms.
In addi ion, in ecen yea s i has been p o en ha non-an ibio ic
compounds, such as coppe , also p omo e an ibio ic esis ance
h ough co-selec ion in e es ial en i onmen s con amina ed
wi h his me al (Li e al., 2017;Pal e al., 2017).
Coppe is c ucial o biological p ocesses and is necessa y
o he su i al o mos li ing o ganisms. I unc ions as a
co ac o o enzymes ha a e in ol ed in elec on ans e ,
oxygen anspo and edox eac ions, and pa icipa es in
impo an p ocesses such as espi a ion, pho osyn hesis, i on
homeos asis, de ense agains oxida i e s ess, and pigmen a ion
(Rensing and McDe i , 2013;Ladome sky and Pe is, 2015).
This essen iali y o coppe is due o i s abili y o oscilla e
be ween wo oxida ion s a es: Cu(I) and Cu(II). Bu his
cha ac e is ic also makes coppe oxic o cells. Coppe , di ec ly
o indi ec ly, gene a es oxygen adicals ha a e esponsible
o lipid pe oxida ion, p o ein oxida ion, damage o nucleic
acids, and des abiliza ion o i on-sul u g oups in many p o eins
(Macombe and Imlay, 2009;Wald on e al., 2009;Dupon
e al., 2011). This dual e ec o coppe means ha bac e ia
mus egula e he in acellula le els o his me al o mee
hei physiological needs and a oid damage. This need o
cellula con ol is also exploi ed by he o ganisms. Fo example,
me al in oxica ion (especially wi h coppe ) is an an imic obial
s a egy used by mac ophages in he immune sys em o educe
he in acellula su i al o pa hogens (Hodgkinson and Pe is,
2012;Djoko e al., 2015;Chand angsu e al., 2017). In a
simila manne , coppe is used in he phagosomes o some
p eda o ameba o kill bac e ia, con ibu ing o p eda ion. These
euka yo ic o ganisms accumula e coppe du ing phagocy osis
by up- egula ing genes encoding p o eins implica ed in coppe
handling and a icking, which inc eases coppe le els in he
phagosome (Hao e al., 2016). Simul aneously, bac e ia espond
o his a ack by up- egula ing he exp ession o genes in ol ed
in coppe de oxi ica ion (Wolschendo e al., 2011;Djoko
e al., 2015). This in e ac ion indica es ha coppe esponse
mechanisms in bac e ia play c ucial oles in pa hogenesis and
esis ance o p eda ion. Howe e , al hough he use o coppe by
mac ophages and euka yo ic p eda o s o kill he p ey and he
esponse o he p ey o elude he damage by his me al ha e
been clea ly es ablished, he ole o coppe in bac e ial p eda ion
emains o be elucida ed.
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
I has ecen ly been epo ed ha he bac e ial li es yle
shapes hei coppe - ela ed p o eome, and his is pa icula ly
obse ed in sys ems in ol ed in coppe homeos asis (An oine
e al., 2019). The soil bac e ium Cup ia idus neca o , which
is a p eda o ex ao dina ily esis an o coppe , uses a oxic
coppe -binding pep ide o kill he ac inomyce e Ag omyces
amosus (Casida, 1987, 1988), and i s p eda o y ac i i y agains
B. sub ilis inc eases a e exposu e o coppe in a concen a ion-
dependen manne (Secca eccia e al., 2016). M. xan hus is he
soil bac e ium wi h he mos complex coppe esponse known
o da e (Pé ez e al., 2018). I s genome codes o a la ge numbe
and wide a ie y o egula o y and s uc u al elemen s in ol ed
in e lux, complexa ion and oxida ion o coppe (Sánchez-Su il
e al., 2007, 2013, 2016;Mo aleda-Muñoz e al., 2010a,b, 2019;
Gómez-San os e al., 2011;Ma cos-To es e al., 2016;Pé ez
e al., 2018). Al hough i has been epo ed ha some o
hese coppe -de oxi ica ion sys ems con ibu e o comple ing he
de elopmen al cycle o his bac e ium upon s a a ion (Sánchez-
Su il e al., 2007, 2013;Ma cos-To es e al., 2016), i is no
clea why his bac e ium encodes such a high numbe o
genes o espond o luc ua ions in coppe concen a ions. One
possibili y could be ha M. xan hus needs his high abundance o
mechanisms ela ed o coppe o su i e p eda ion by p o ozoa
in soils. Howe e , i is also easonable o hink ha M. xan hus
may u ilize coppe as pa o i s a senal o poison and kill
bac e ial p ey, in a simila manne o euka yo ic p eda o s and
mac ophages. In his s udy, we explo e his possibili y by using
S. melilo i, an ag icul u ally impo an ni ogen- ixing bac e ium
ha con ibu es o he e ili y o soils, as a p ey. Ou esul s
e eal ha coppe accumula es in he p eda o y c ossing poin ,
whe e he wo bac e ia come in o con ac , up- egula ing he
coppe de oxi ica ion mechanisms in he p eda o , and igge ing
he o e p oduc ion o melanin in he p ey o de end i om
p eda ion. These esul s indica e ha coppe also plays a ole in
bac e ial p eda ion.
MATERIALS AND METHODS
Bac e ial S ains, Plasmids, and G ow h
Condi ions
Geno ypes o he E. coli,M. xan hus, and S. melilo i s ains,
plasmids, and oligonucleo ides used in his s udy a e lis ed
in Supplemen a y Tables S1–S3, espec i ely. E. coli s ains
we e g own in lysogenic b o h (Samb ook and Russell, 2001) a
37◦C, supplemen ed wi h kanamycin (25 µg/ml) when necessa y.
M. xan hus s ains we e g own in CTT b o h (Hagen e al., 1978)
a 30◦C wi h igo ous shaking (300 pm). When needed, X-gal (5-
b omo-4-chlo o-3-indolyl-β-D-galac opy anoside, 100 µg/ml),
kanamycin (80 µg/ml), and/o di e en me als [CuSO4·
7H2O, Cd(NO3)2·4H2O, o Zn(NO3)2·6H2O] we e added o
he medium a he concen a ions indica ed in each igu e.
S. melilo i s ains we e g own a 30◦C in CTT b o h, TY
complex medium (Be inge , 1974) o de ined minimal medium
MM (Robe sen e al., 1981), supplemen ed wi h kanamycin
(200 µg/ml), chlo amphenicol (25 µg/ml), suc ose (10%), and/o
he concen a ion o me als [CuSO4·7H2O, Cd(NO3)2·4H2O,
o Zn(NO3)2·6H2O] indica ed in each igu e, when necessa y.
Se e al o he bac e ia we e used o examine coppe accumula ion
in he p eda o y in e ace wi h M. xan hus. All o hem we e
g own in lysogenic b o h. These bac e ia we e ob ained om
he ‘Colección del Depa amen o de Mic obiología’ (Uni e sidad
de G anada, Spain) (Bacillus la e ospo us,B. sub ilis,E. coli,
Mic ococcus sp., Salmonella sp., S aphylococcus au eus, and
P o eus sp.), Fo solid media, Bac o-Aga (Di co) was added a
a concen a ion o 1.5%.
Nucleic Acid Manipula ions
Fo nucleic acid manipula ions, ou ine molecula biology
echniques we e used (Samb ook and Russell, 2001). Plasmids
we e in oduced in o E. coli by ans o ma ion (Samb ook and
Russell, 2001), in o M. xan hus by elec opo a ion (Kashe i
and Ha zell, 1995), and in o S. melilo i by conjuga ion
(Simon e al., 1983).
P eda ion Expe imen s
Fo hese expe imen s, all M. xan hus and S. melilo i s ains
we e g own in CTT b o h and TY b o h (wi h an app op ia e
an ibio ic, when necessa y), espec i ely, wi h igo ous shaking
a 30◦C o an op ical densi y a 600 nm (OD600) o 1. Cells we e
cen i uged and concen a ed in TM bu e (10 mM T is-HCl
[pH 7.6], 1 mM MgSO4) o a inal OD600 o 15 o M. xan hus
and 5 o S. melilo i s ains. D ops o 10 µl o he S. melilo i
suspensions we e deposi ed on o he su ace o CTT aga pla es
and allowed o d y. Nex , d ops o 10 µl o he a ious M. xan hus
suspensions we e spo ed in close p oximi y o he S. melilo i spo
(no mo e han 1 mm sepa a ion be ween spo s). Pla es om 9
eplica es o each condi ion we e incuba ed a 30◦C and images
om ep esen a i e samples we e aken wi h a digi al came a and
an Olympus SZX7 dissec ing mic oscope.
Fo cell coun ing and de e mining he iabili y o he p ey,
M. xan hus and S. melilo i s ains we e g own as indica ed abo e.
Howe e , o his pu pose hizobial cul u es we e dilu ed o a
inal OD600 o 0.2. D ops o 10 µl we e deposi ed on o memb ane
il e s (Isopo e, Millipo e) placed on he su ace o CTT aga
pla es and incuba ed a 30◦C o 24 h. A his ime, some il e s
we e collec ed as desc ibed below o ob ain he numbe o cells
a 0 h. In he es o he pla es, 10 µl o concen a ed cul u e
a an OD600 o 15 o he M. xan hus DK1622 wild- ype s ain
we e deposi ed on op o he hizobial colonies. A e 24 h
o incuba ion, he il e s we e deposi ed in an eppendo ube,
apped wi h ube s oppe s and ho oughly washed wi h 1 ml
o TM bu e . A e cen i uga ion o he il e s o 3 min a
12000 pm, he supe na an s and il e s we e disca ded, and he
pelle s esuspended in 500 µl o TM bu e . Viable S. melilo i
cells we e coun ed by using he dilu ion me hod on o TY aga
supplemen ed wi h chlo amphenicol, which allows g ow h o
S. melilo i s ains while inhibi ing M. xan hus g ow h. The
esul s a e he a e age o h ee independen biological eplica es.
S a is ical compa isons be ween s ains we e pe o med using
S uden ’s - es . Di e ences we e conside ed signi ican a a le el
o p<0.05.
Fo melanin de e mina ions, we used he same p ocedu e
men ioned abo e, excep ha cells we e no spo ed on op
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
o a il e . A e incuba ion, cells we e di ec ly sc aped om
he cul u e media.
Cons uc ion o S ains Ha bo ing lacZ
Fusions and β-Galac osidase Assays
Myxococcus xan hus DK1622 s ain was elec opo a ed wi h
plasmids ha bo ing ansc ip ional usions be ween he genes
o in e es o his myxobac e ium (cuoA,copA,cus2, and czc3)
and lacZ om E. coli (Supplemen a y Table S2) o gene a e he
kanamycin- esis an ans o man s (Supplemen a y Table S1),
which we e con i med by Sou he n blo analysis. Fo quali a i e
β-galac osidase ac i i y analyses du ing p eda ion, 10-µl d ops o
M. xan hus and S. melilo i suspensions a an OD600 o 15 and 5
espec i ely we e ob ained as indica ed abo e and spo ed in close
p oximi y on o CTT aga pla es supplemen ed wi h X-gal and he
me als indica ed in he igu es. Pla es om 9 eplica es o each
condi ion we e incuba ed a 30◦C. Blue colo de elopmen as a
esul o β-galac osidase ac i i y in ep esen a i e samples was
obse ed wi h an SZX7 Olympus dissec ing mic oscope.
Cons uc ion o S. melilo i In-F ame
Dele ion Mu an s
To gene a e he S. melilo i GR4 mepA and mcoA dele ion
mu an s, ORF lanking sequences (736-bp ups eam and 855-
bp downs eam o mepA, and 782-bp ups eam and 799-bp
downs eam o mcoA) we e PCR ampli ied by Phusion High-
Fideli y DNA Polyme ase (The mo Fishe Scien i ic) using
S. melilo i GR4 genomic DNA as a empla e and he p ime s
lis ed in Supplemen a y Table S3. Fo he double dele ion
mu an , sequences 782-bp ups eam and 832-bp downs eam o
he mcoA gene we e PCR ampli ied using S. melilo i mu an
1mepA genomic DNA as a empla e and he app op ia e
p ime s (Supplemen a y Table S3). Pai ed PCR agmen s,
ca ying an in e nal BamHI (o XmaI o he double mu an )
and an ex e nal EcoRI/XbaI es ic ion si e, we e inse ed in o
he ec o pK18mobsacB, yielding he plasmids pK181mepA,
pK181GR4pB023 and pK181GR4pB023.2 (Supplemen a y
Table S2), which we e mobilized in o he wild- ype GR4 s ain
(o 1mepA) om E. coli by conjuga ion.
T ansconjugan s a ising om a single c oss-o e e en
we e selec ed as kanamycin- esis an colonies in MM
medium (and simul aneously e i ied o suc ose sensi i i y
in TY aga supplemen ed wi h 10% suc ose). Kanamycin-
esis an /suc ose-sensi i e colonies we e s eaked on
suc ose-supplemen ed TY aga pla es o selec double
c oss-o e e en s. The dele ion o genes in he colonies
hus ob ained was checked by PCR using he p ime s
mepA_E o /mepA_X e and SmMCOE o /SmMCOX e (o
XSmMCO e 2) (Supplemen a y Table S3), ollowed by BamHI
(o XmaI) es ic ion o he PCR p oduc s and sequencing.
UV-Visible (UV-V) Spec opho ome ic
Analysis o Ex ac s Con aining
Melanin-Type Pigmen s
Pigmen s om S. melilo i g own as pu e cul u es o in co-
cul u e wi h M. xan hus (as indica ed abo e o cell coun ing and
iabili y de e mina ions) on CTT aga pla es in he p esence o
50 µM coppe we e ex ac ed ollowing a modi ied e sion o
he p o ocol desc ibed by Whi ake (1963). B ie ly, cells we e
pelle ed in an eppendo ube and solubilized in 1 ml o 1 N
NaOH and 10% dime hyl sul oxide o 2 h a 80◦C. Nex , samples
we e cen i uged a 12000 g o 10 min a oom empe a u e, and
supe na an s we e ans e ed o esh ubes. Samples we e hen
scanned in a UV-V spec opho ome e (Ca y 50 Conc, Va ian) a
UV and isible wa eleng hs (230–600 nm). The blank con ol was
1 N NaOH solu ion.
Pu i ica ion and Fou ie T ans o m
In a ed (FT-IR) Spec oscopy o he
Melanin-Type Pigmen
Fo hese analyses, pigmen s we e pu i ied using he me hods
desc ibed by Sajjan e al. (2010) and D ewnowska e al. (2015).
B ie ly, solu ions con aining he pigmen s ex ac ed as indica ed
in he p e ious sec ion we e acidi ied by lowe ing he pH o 2.0
using 1 N HCl and incuba ed a oom empe a u e o 1 week,
ollowed by boiling o he suspension o 1 h. P ecipi a es we e
hen collec ed by cen i uga ion a 14000 g o 15 min, and pelle s
we e washed h ee imes wi h 0.1 N HCl and once wi h double-
dis illed wa e . Nex , pelle s we e esuspended in 10 ml e hanol
and suspensions we e boiled o 10 min and incuba ed a oom
empe a u e o 1 day. Then, samples we e cen i uged as abo e
and pelle s we e washed wice wi h e hanol. Finally, pu i ied
pigmen s we e allowed o d y a oom empe a u e and s o ed
a −20◦C un il u he use.
The FT-IR spec a o he pu i ied pigmen s we e eco ded by
a JASCO 6200 in a ed spec opho ome e . Samples we e placed
on a diamond window o he spec ome e , and measu emen s
we e done in e lec ion mode, a oom empe a u e, by a e aging
32 scans wi h a esolu ion o 2 cm−1in he spec a ange
400–4000 cm−1.
Mic oscopy S udies
Va iable P essu e Scanning Elec on Mic oscopy
(VPSEM)
Fo scanning elec on mic oscopy (SEM), cells om 24-h co-
cul u es o M. xan hus and S. melilo i on CTT aga pla es we e
ixed wi h glu a aldehyde apo s o 24 h a oom empe a u e.
Then, samples we e washed h ee imes (5 min each) in 0.1 M
cacodyla e bu e . Dehyd a ion was accomplished by a g aded
se ies o e hanol. Samples we e hen c i ical-poin d ied and
spu e -coa ed wi h gold.
The in e ace be ween M. xan hus and S. melilo i was analyzed
by ene gy dispe si e X ay (EDX), imaging seconda y elec ons
(SE) and backsca e ed elec ons (BSE) in a FESEM Zeiss Sup a
40Vp equipped wi h an Az ec 2.1 XMax sys em. Mic oanalyses
we e ca ied ou a 20 kV.
High-Resolu ion T ansmission Elec on Mic oscopy
(HRTEM)
Samples, aken om he c ossing poin and om he dis al edges
o M. xan hus and S. melilo i, we e ixed as desc ibed abo e, and
hen pos ixed wi h osmium e oxide. Images and quali a i e
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
analysis we e ob ained in a high- esolu ion ansmission elec on
mic oscope FEI TITAN G2 a an accele a ion ol age o 300 kV,
wi h a sphe ical abe a ion co ec o and high-angle annula da k
ield (HAADF) ype de ec o , which p oduces an annula da k
ield image. Resolu ion in ansmission mode was 0.8 Å and in
scanning ansmission elec on mic oscopy (STEM) 1.3 Å. The
mic oscope is equipped wi h an XFEG ype cannon and a Supe X
EDX mic oanalysis sys em.
RESULTS AND DISCUSSION
Coppe Helps M. xan hus o Pene a e
he S. melilo i Colony and Induces he
P oduc ion o a B own Pigmen in he
P ey
To in es iga e he ole o coppe in M. xan hus p eda ion, he
non-mucoid wild- ype s ain Rm1021 o S. melilo i was es ed
as a p ey by spo ing d ops o p eda o and p ey nex o each
o he on solid media (see sec ion “Ma e ials and Me hods”). Two
coppe concen a ions ha a e p esen e en in non-con amina ed
soils we e assayed (Sánchez-Su il e al., 2007). As obse ed
in Figu e 1,M. xan hus ad anced u he h ough he p ey
colonies as he coppe concen a ion inc eased, indica ing ha
his me al acili a es he pene a ion o he p eda o . Mo eo e ,
i was also obse ed ha he yellow colo o he M. xan hus
colonies emained nea ly unal e ed by he coppe concen a ions
used in hese assays. In con as , a b own colo was obse ed
in he S. melilo i s ain when cells we e incuba ed in media
wi h coppe , which was da ke a highe concen a ions o he
me al in bo h he pu e cul u e and co-cul u e o p ey and
FIGURE 1 | P eda o y ac i i y o M. xan hus DK1622 e sus S. melilo i
Rm1021 in he p esence o di e en coppe concen a ions and in he
absence o his me al. Pic u es we e aken a 72 h unde a dissec ing
mic oscope wi h illumina ion om he op o he colonies.
p eda o (Figu e 1). Howe e , his colo was no obse ed in
media wi hou coppe . Fu he mo e, he b own colo was much
da ke in he a ea whe e p ey and p eda o coexis han in he
a ea we e he p ey has no ye been eached by he p eda o .
Al hough i migh be hough ha his colo could o igina e
om he supe posi ion o bo h bac e ia, his does no seem
o be he case, since he colo emains yellow in his egion
when no coppe is added o he medium (Figu e 1). These
esul s indica e ha coppe acili a es M. xan hus p eda ion and
induces he o e p oduc ion o a pigmen in he p ey, mos likely
o de end i om he p eda o a ack. Since his pigmen is
p oduced in highe amoun s in he p esence o coppe (see panels
in Figu e 1 wi h only hizobia), his obse a ion sugges s ha
his me al accumula es in he egion whe e p eda o and p ey
o e lap. An explana ion o why M. xan hus pene a es he p ey
colonies mo e easily in he p esence o coppe could be ha
hese coppe concen a ions s imula e g ow h. Howe e , i has
been epo ed ha g ow h a es o M. xan hus emains nea ly
cons an wi h coppe concen a ions be ween 0 and 500 µM
coppe (Sánchez-Su il e al., 2007;Gómez-San os e al., 2012). In
con as , ad en u ous and social mo ili y in his myxobac e ium
a e s imula ed by he coppe concen a ions used in his s udy
(Gómez-San os e al., 2012). Taking in o conside a ion ha bo h
ypes o mo ili y a e equi ed o p ope p eda ion, since mu an s
in any mo ili y sys em p ey wi h h ee o de o magni ude less
e icien ly han he wild- ype s ain on S. melilo i Rm1021 (Pé ez
e al., 2014), s imula ion o A and S mo ili y by coppe migh
acili a e pene a ion o M. xan hus on he p ey colony.
Coppe Accumula es in he P eda o y
C ossing Poin
Se e al ypes o analyses we e ca ied ou o ind ou
whe he coppe accumula es in he egion whe e p eda o and
p ey collide. Fi s , se e al s ains ha bo ing usions be ween
p omo e s o me al- esponsi e and non- esponsi e genes and
lacZ we e cons uc ed in M. xan hus DK1622 o be used
as biosenso s (Supplemen a y Table S1). The genes chosen
o hese analyses we e cuoA,copA,cus2, and czc3 (Sánchez-
Su il e al., 2007;Mo aleda-Muñoz e al., 2010a,b). cuoA and
copA encode a mul icoppe oxidase (MCO) and a P1B- ype
ATPase espec i ely, whose exp essions ha e been epo ed o
be dependen on coppe and o he di alen me als, such as
zinc (Sánchez-Su il e al., 2007;Mo aleda-Muñoz e al., 2010b).
In con as , he sys ems Cus2 and Czc3 a e bo h ipa i e
complexes in ol ed in hea y me al e lux. Howe e , while he
exp ession o genes o he Cus2 sys em is dependen on coppe
and cadmium, genes o he Czc3 sys em exhibi a cons i u i e
me al-independen exp ession. The usion cons uc ed in he
Czc3 sys em was used as a nega i e con ol (Mo aleda-Muñoz
e al., 2010a). The biosenso s ains and S. melilo i Rm1021 we e
hen spo ed on o CTT aga pla es con aining X-gal and di e en
coppe and o he me al concen a ions o isualize he blue colo
de elopmen in di e en egions o he co-cul u e. As shown in
he pho og aphs on he le o Figu e 2A, cells ha bo ing usions
wi h he genes cuoA,copA and cus2 incuba ed in he p esence
o coppe ha glide om he o iginal spo o a egion whe e he
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
FIGURE 2 | De ec ion o coppe accumula ion in he p eda o y in e ace o M. xan hus and S. melilo i by using M. xan hus coppe biosenso s s ains. (A) M. xan hus
s ains ha bo ing usions be ween genes cuoA,cus2 and copA, and lacZ we e co-cul u ed wi h S. melilo i Rm1021 in he p esence o coppe . (B) As nega i e
con ols, M. xan hus s ains ha bo ing usions be ween genes czc3,cuoA, and cus2, and lacZ we e co-cul u ed wi h S. melilo i Rm1021 in he absence o me als,
zinc, and cadmium, espec i ely. S ains we e spo ed on o CTT aga pla es con aining 100 µg/ml X-gal o isualize he blue colo de elopmen . Pic u es we e aken
a e 48 o 72 h o incuba ion unde a dissec ing mic oscope wi h illumina ion om he bo om o he colonies.
p ey is no p esen de elop a ligh e blue colo , p obably as a
esul o lowe densi y and sho e ime o hyd olyze X-gal. In
con as , when hey app oach and en e he p ey colony, he blue
colo is much da ke , and simila o ha obse ed in he cen e
o he o iginal spo (Figu e 2A, igh pho og aphs). I could
be a gued ha he cell densi y o he p eda o in he p eda o -
p ey in e ace is highe han in o he su ounding egions due
o he consump ion o he p ey. Howe e , he in ensi y o he
blue colo is qui e simila in all he egions a ound he o iginal
spo in he case o he M. xan hus s ain ha bo ing he usion
in he cons i u i e czc3 sys em (compa e pho og aphs on le
and igh o Figu e 2B, uppe pa ), indica ing ha his does
no seem o be he case. Mo eo e , blue colo accumula ion
was no de ec ed a he c ossing zones when s ains ha bo ing
usions in he cuoA and cus2 genes we e incuba ed in he
p esence o zinc and cadmium espec i ely (Figu e 2B, compa e
pho og aphs on le and igh whe e hese wo s ains a e
shown), in spi e o he ac ha hese genes a e also up-
egula ed by hese me als (Sánchez-Su il e al., 2007;Mo aleda-
Muñoz e al., 2010a). These obse a ions seem o indica e
ha coppe accumula es du ing p eda ion whe e M. xan hus
makes con ac wi h S. melilo i. Coppe accumula ion was also
obse ed in he p eda o y in e ace o M. xan hus wi h o he
G am-nega i e (P o eus,Salmonella, and E. coli) and G am-
posi i e (Mic ococcus) bac e ia. Howe e , du ing he in e ac ion
o M. xan hus wi h o he G am-posi i e bac e ia blue colo
accumula ion was no appa en (B. la e ospo us and S. au eus)
o was absen (B. sub ilis) (Supplemen a y Figu e S2), indica ing
ha coppe accumula es only in some in e ac ions p eda o -p ey.
A second me hod o de e mining whe he coppe
accumula es in he egion whe e p eda o and p ey a e in
con ac was SEM coupled wi h EDX. This me hod was applied
in he a ea o con ac (Figu e 3A, delinea ed in blue), and a
segmen (d awn in g een in Figu es 3A,B) was chosen o he
scan mic oanalysis o di e en chemical elemen s. Ca bon and
oxygen we e analyzed o de e mine he quan i y o cells in his
a ea. As can be obse ed in Figu e 3B, hese wo elemen s
exhibi non-linea p o iles, as expec ed om he s acking o
bac e ia when he wo on s collide, which is obse ed in
Figu e 3A. Howe e , coppe exhibi ed a di e en p o ile, since
he amoun o his me al inc eases as M. xan hus app oaches
and makes con ac wi h he p ey, hen dec eases a g ea e
dis ances, whe e mo e p ey and less p eda o a e p esen . Finally,
o u he in es iga e whe he coppe accumula es a he c ossing
poin , samples om he dis al edges o he p eda o and p ey,
and om he in e ace zone, we e p epa ed and examined
by using HRTEM coupled o an EDX mic oanalysis sys em.
As obse ed in Figu e 3C, he coppe signal (images in ed)
was o highe in ensi y a he c ossing poin han a he dis al
edges, whe e only p ey o p eda o a e p esen . These esul s
co obo a e he accumula ion o coppe a he con ac poin as a
consequence o in e ac ion.
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
FIGURE 3 | De e mina ion o coppe by elec on mic oscopy a he in e ac ion c ossing poin o M. xan hus and S. melilo i in he p esence o 10 µM coppe .
(A) Pic u es o he egion whe e p eda o and p ey collide we e aken wi h a dissec ing mic oscope (le pic u e), a a iable p essu e scanning elec on mic oscope
(VPSEM), and a VPSEM equipped wi h an elec on dispe si e X ay (EDX). Pic u es o EDX we e aken by backsca e ed elec ons (BSE), which ca ies in o ma ion
abou he composi ion o he sample, and seconda y elec ons (SE), which inspec s he opog aphy o he sample su ace. Squa es indica e he sec ion used o
p epa e he samples o whe e he di e en image ampli ica ions we e aken. (B) Semi-quan i ica ion o ca bon, oxygen and coppe by using VPSEM in he selec ed
sec ion (g een segmen ) ep esen ed in he uppe pho og aph aken wi h EDX. (C) High esolu ion ansmission mic oscopy wi h EDX mic oanalysis o coppe (black
and ed pic u es) in he egions shown wi h squa es in he image aken unde a dissec ing mic oscope (cen al pic u e). Black and whi e images o he same a ea
we e aken wi h a high esolu ion scanning ansmission elec on mic oscope using a HAADF de ec o . Samples o mic oscopy p epa a ions we e aken a 48 h.
The esul s ob ained wi h coppe biosenso s and EDX
mic oanalyses indica e ha coppe accumula es in he egion
whe e he p eda o and p ey make con ac , and sugges ha
he e mus be an al e a ion o coppe a icking in he p eda o y
in e sec ion zone.
S. melilo i O e p oduces Melanin in
Response o he In asion by M. xan hus
As men ioned abo e, a b own pigmen is p oduced by S. melilo i
Rm1021 in he p esence o coppe , exhibi ing a highe in ensi y
in he egion whe e he p ey and p eda o coexis (Figu e 1).
We had se e al easons o suspec ha his pigmen migh
be he a oma ic polyme melanin. (1) The p oduc ion o a
melanin-like pigmen has been p e iously epo ed in se e al
hizobia (Cubo e al., 1988), including S. melilo i GR4, whose
exp ession is dependen on coppe (Me cado-Blanco e al., 1993;
Cas o-Sowinski e al., 2002). (2) An impo an biological ole
in oked o melanins is me al chela ion (Hong and Simon,
2007), including coppe , which is seques e ed by his polyme
o p o ec cells om oxida i e s ess (Ko y owski e al., 1985;
Hamil on and Gomez, 2002). (3) Melanins a e also in ol ed
in esis ance o a ack by di e en cell-wall enzymes (Kuo and
Alexande , 1967;Rosas and Casade all, 2001;S eenbe gen e al.,
2001), imp o ing su i al and compe i i e skills unde se e al
en i onmen al s esses (Bell and Wheele , 1986;Coyne and Al-
Ha hi, 1992;Nosanchuk and Casade all, 2003;Zhang e al.,
2007). These p e ious s udies p omp ed us o de e mine whe he
he b own pigmen p oduced by S. melilo i in he p esence
o coppe , and especially a he p eda o -p ey in e ace, could
be melanin, which would unc ion as a de ensi e mechanism
agains p eda ion.
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
Me cado-Blanco e al. (1993) epo ed ha S. melilo i GR4
p oduced melanin in he p esence o coppe and y osine,
and demons a ed ha when he gene o he y osinase
MepA, encoded in he plasmid pRmeGR4b, was exp essed in
Ag obac e ium ume aciens and E. coli, hese bac e ia p oduced
melanin. Since no s udies abou melanin biosyn hesis a e
a ailable o he s ain Rm1021, o de e mine whe he he b own
pigmen obse ed in he co-cul u e o M. xan hus and S. melilo i
in he p esence o coppe was his complex and a ied phenolic
polyme , we decided o use he GR4 s ain o S. melilo i ins ead o
Rm1021 as he p ey. Fi s , we checked whe he he s ain GR4
beha ed like he s ain Rm1021. The esul s ob ained e ealed
ha M. xan hus pene a ed mo e easily in he colony o his
hizobial s ain, ha he GR4 s ain p oduced a b own pigmen
in he p esence o coppe , wi h highes in ensi y a he p ey-
p eda o in e ace, and ha he biosenso s ains o M. xan hus
ha bo ing usions be ween he coppe -inducible genes copA,
cuoA and cus2, and lacZ also exhibi ed he highes β-galac osidase
ac i i y in he a ea whe e hey collide wi h S. melilo i GR4
(Supplemen a y Figu e S1).
Nex , pigmen was ex ac ed om S. melilo i GR4 g own on
pu e cul u e and co-cul u e wi h M. xan hus in he p esence
o coppe a e 48 h o incuba ion. Ex ac s we e hen analyzed
by FT-IR spec oscopy. As shown in Figu es 4A,B, he FT-IR
spec a ob ained o he solid b own pigmen ex ac ed in bo h
samples we e e y simila . Mo eo e , hese spec a exhibi ed
se e al ea u es ha a e iden ical o hose desc ibed o bac e ial
melanins o E. coli (Chá ez-Béja e al., 2013;Mejia-Caballe o
e al., 2016), Lysobac e oligo ophicus (Kimu a e al., 2014),
FIGURE 4 | Analysis o he b own pigmen p oduced by S. melilo i GR4 in he
p esence o coppe . FT-IR (A,B) and UV-V (C,D) spec a o he d y pigmen
ex ac ed om pu e cul u es o S. melilo i GR4 (blue ec angle) and
co-cul u es wi h M. xan hus ( ed ec angle). Ex ac s we e ob ained a e 48 h
o incuba ion on CTT aga pla es supplemen ed wi h 50 µM coppe .
Azo obac e ch oococcum (Bane jee e al., 2014), Pseudomonas
(Ta angini and Mish a, 2013), S ep omyces cyaneo usca us (Al
Kha ib e al., 2018), Bacillus weihens ephanensis (D ewnowska
e al., 2015), and soil bac e ia (Ta angini and Mish a, 2014).
Ne e heless, o u he con i m ha he pigmen was melanin,
he abso bance in UV-V o pigmen s ex ac ed in NaOH om a
pu e cul u e o S. melilo i GR4 and a co-cul u e wi h M. xan hus
ollowing a modi ied e sion o he p o ocol desc ibed by
Whi ake (1963) (see sec ion “Ma e ials and Me hods” o
de ails) was also de e mined. As shown in Figu es 4C,D, he
spec a ob ained esemble hose desc ibed o o he melanins
(Szpoganicz e al., 2002), exhibi ing a peak a 243 nm. In
he ex ac s om he co-cul u e o bo h bac e ia, addi ional
peaks we e de ec ed wi h maxima a 386–405 nm (Figu e 4D).
Those peaks a e mos likely due o he M. xan hus yellow
pigmen DKxan hene, which belongs o a amily o seconda y
me aboli es ha shows maxima o abso p ion a ound 400 nm
(Meise e al., 2006).
Iden i ica ion o S. melilo i GR4 Genes
In ol ed in Melanin Biosyn hesis
Sino hizobium melilo i GR4 (Casadesús and Oli a es, 1979) holds
a genome wi h a o al leng h o 7.15 Mb (Ma ínez-Aba ca e al.,
2013), comp ising he ch omosome (3.62 Mb), wo small s able
plasmids, pRmeGR4a (0.18 Mb) and pRmeGR4b (0.23 Mb),
and wo pSym plasmids, pRmeGR4c (1.42 Mb) and pRmeGR4d
(1.70 Mb). As men ioned abo e, Me cado-Blanco e al. (1993)
demons a ed ha mepA encodes a y osinase in ol ed in
melanin biosyn hesis, which is included in plasmid pRmeGR4b.
Howe e , aking in o conside a ion ha in hei expe imen s
hey used 10 imes as much coppe as we did (25 µM coppe
e sus 250 µM), ha hey needed o supplemen he cul u e
media wi h y osine (which was no included in ou assays),
and ha hey used pu e cul u es while we obse ed he highes
in ensi y o b own colo in he co-cul u e o S. melilo i wi h
M. xan hus, we decided o ca y ou a numbe o expe imen s
o iden i y he genes ha a e in ol ed in melanin biosyn hesis
when he hizobium has o de end i sel om he p eda o . Fi s ,
M. xan hus was assayed agains he s ains o S. melilo i GRM8SR
and GRM10, bo h de i ed om GR4 (Me cado-Blanco e al.,
1993). GRM10 possesses he plasmids pRmeGR4b, pRmeGR4c
and pRmeGR4d, while GRM8SR only con ains he plasmids
pRmeGR4c and pRmeGR4d (Supplemen a y Table S1). As can
be obse ed in Figu e 5, he s ains GR4 and GRM10 syn hesize
melanin, and only in he s ain GRM8SR is he da k pigmen no
obse ed. The same esul is obse ed in pu e cul u es and co-
cul u es, indica ing ha he melanin biosyn he ic enzymes ha
a e up- egula ed du ing p eda ion in he p esence o coppe a e
encoded in he non-symbio ic plasmid pRmeGR4b, whe e he
gene mepA is loca ed (Me cado-Blanco e al., 1993).
Me cado-Blanco e al. (1993) demons a ed he y osinase
ac i i y o mepA by exp essing i in E. coli and analyzing he
ex ac s in non-dena u ing gels. To s udy he ole o MepA in
melanin biosyn hesis in S. melilo i and in p o ec ion agains
p eda ion, an in- ame dele ion 1mepA mu an was cons uc ed
(Supplemen a y Table S1). The analysis o his mu an o
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Con e as-Mo eno e al. Coppe and Myxococcus xan hus P eda ion
FIGURE 5 | Melanin p oduc ion by S. melilo i GR4 and i s de i a i es GRM10 and GRM8SR s ains in he absence and he p esence o coppe in pu e cul u es and
co-cul u e wi h M. xan hus. Cells we e incuba ed o 72 h. Then, 30 µl o SDS a 10% was added o he colonies, and pla es we e incuba ed o 24 h a oom
empe a u e p io o aking pho og aphs unde a dissec ing mic oscope wi h illumina ion om he op o he colonies.
melanin p oduc ion e ealed ha he amoun o SDS-ex ac able
b own pigmen was much smalle han in he wild- ype s ain
(Figu e 6). Howe e , he colo o he mu an was sligh ly da ke
han ha obse ed in he GRM8SR s ain, indica ing ha o he
gene(s) mus emain in he pRmeGR4b plasmid wi h he abili y
o syn hesize melanin. To iden i y o he genes in his plasmid
in ol ed in he p oduc ion o melanin, a bioin o ma ic analysis o
he plasmid sequence was ca ied ou (Supplemen a y Table S4).
Sequences o all open eading ames included in his plasmid
(accession numbe CP003935; Ma ínez-Aba ca e al., 2013)
we e eanalyzed, sea ching o domains in he P am da abase
(Finn e al., 2015), unc ions o he p o eins and me abolic
pa hways in he KEGG da abase (Kanehisa and Go o, 2000),
and BLAST analyses in he NCBI da abase1. The mos plausible
candida e o be in ol ed in melanin biosyn hesis was he gene
C770_GR4pB023 (designa ed mcoA), which encodes an MCO
wi h he domains Cu-oxidase_3 and Cu-oxidase_2 in he P am
da abase, and he ou coppe -binding domains ypical o MCOs
(Cha and Cooksey, 1991;B own e al., 1995;Ou en e al., 2000;
Sánchez-Su il e al., 2007). Some MCOs exhibi laccase ac i i y
and a e able o syn hesize melanin in ungi and bac e ia (Hullo
e al., 2001;Cas o-Sowinski e al., 2002, 2007;Ma ins e al.,
2002;He nández-Rome o e al., 2005;Sapmak e al., 2015;Li
e al., 2016). Ou analyses also e ealed ha his gene o ms a
clus e wi h a hypo he ical p o ein (C770_GR4pB024) and mepA
(C770_GR4pB025). These h ee genes conse e mic osyn eny
1h p://blas .ncbi.nlm.nih.go /Blas .cgi
(acco ding o KEGG da abase) wi h o he S. melilo i s ains, such
as BL255C, AK83 and SM1. Syn eny is also conse ed wi h o he
Sino hizobium s ains and ela ed gene a (S. medicae and S. edii
HH10, B ady hizobium sp. CCGE-LA001, Meso hizobium cice i,
M.oppo unis um and M. aus alicum), and in o he non- ela ed
bac e ia such as Caulobac e la us and G anulicella mallensis.
The conse a ion o he genomic a chi ec u e sugges s ha hese
h ee genes a e implica ed in he same unc ion.
To in es iga e he ole o his MCO in melanin
biosyn hesis and p eda ion, a 1mcoA mu an was cons uc ed
(Supplemen a y Table S1), and he analysis o he SDS-
ex ac able pigmen in he gene a ed mu an e ealed ha
dele ion o his gene also diminishes melanin p oduc ion, as
he 1mepA mu an (Figu e 6). This obse a ion led us o
cons uc a double mu an 1mepA1mcoA, and he analysis o
his s ain indica ed ha i is non-melanogenic, as he s ain
GRM8SR. The e o e, i can be concluded ha bo h McoA and
MepA a e esponsible o melanin biosyn hesis in S. melilo i
GR4 (Figu e 6).
Melanin Plays a De ensi e Role Agains
P eda ion
To de e mine he ole o S. melilo i melanin as a de ensi e
mechanism agains p eda ion, he numbe o cells ha su i e
he p eda o y ac i i y o M. xan hus was de e mined using he
wild- ype s ain GR4, he cu a ed s ain GRM8SR, and he h ee
mu an s gene a ed, 1mepA,1mcoA, and 1mepA1mcoA, as
F on ie s in Mic obiology | www. on ie sin.o g 9Feb ua y 2020 | Volume 11 | A icle 94
