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Exploi ing he biological esponse
o wo Se a ia on icola s ains
o he c i ical me als, gallium
and indium
Joana B. Caldei a, Paula V. Mo ais & Ri a B anco*
The use o mic oo ganisms ha allows he eco e y o c i ical high- ech elemen s such as gallium
(Ga) and indium (In) has been conside ed an excellen eco-s a egy. In his pe spec i e, i is ele an
o unde s and he s a egies o Ga and In esis an s ains o cope wi h hese c i ical me als. This
s udy aimed o explo e he e ec o hese me als on wo Ga/In esis an s ains and o sc u inize he
biological p ocesses behind he oxida i e s ess in esponse o exposu e o hese c i ical me als. Two
s ains o Se a ia on icola, A3242 and B2A1Ga1, wi h high esis ance o Ga and In, we e submi ed
o me al s ess and hei p o ein p o iles showed an o e exp essed Supe oxide Dismu ase (SOD)
in p esence o In. Resul s o inhibi o -p o ein na i e gel incuba ions iden i ied he o e exp essed
enzyme as a Fe-SOD. Bo h s ains exhibi ed a huge inc ease o oxida i e s ess when exposed o
indium, isible by an ex eme high amoun o eac i e oxygen species (ROS) p oduc ion. The oxici y
induced by indium igge ed biological mechanisms o s ess con ol namely, he dec ease in educed
glu a hione/ o al glu a hione le els and an inc ease in he SOD ac i i y. The e ec o gallium in cells
was no so bois e ous, isible only by he dec ease o educed glu a hione le els. Analysis o he
cellula me abolic iabili y e ealed ha each s ain was a ec ed di e en ly by he c i ical me als,
which could be ela ed o he dis inc me al up akes. S ain A3242 accumula ed mo e Ga and In in
compa ison o s ain B2A1Ga1, and showed lowe me abolic ac i i y. Unde s anding he biological
esponse o he wo me al esis an s ains o S. on icola o s ess induced by Ga and In will ackle he
cu en gap o in o ma ion ela ed wi h bac e ia-c i ical me als in e ac ions.
Indium (In) and gallium (Ga) a e me als belonging o g oup 13 on he pe iodic able1 ha ha e di e se appli-
ca ions on he ae ospace, elecommunica ion and in o ma ics indus ies. Gallium is used in a wide a ie y o
p oduc s as gallium a senide (GaAs) o gallium ni ide (GaN). These compounds a e used in he manu ac u e o
sola cells, LEDs, lase s and in he p oduc ion o highly specialized ci cui s ha a e essen ial in high-pe o mance
compu e s and cell phones2. The main applica ions o indium a e he p oduc ion o LCDs and ouch sc eens and
i is also used in he manu ac u e o LEDs and some medical ma e ials3. Face on he economic, indus ial and
echnological impo ance o bo h me als and hei low abundance compa a i ely wi h o he elemen s and me -
als, Ga and In a e included in he c i ical aw ma e ials lis 4. Wi h he inc ease consume o hese c i ical me als,
he con inuous ex ac ion o hese me als om mine o es, seconda y mine al deposi s o disca ded ma e ials
is necessa y and consequen ly, he en i onmen is o en exposed o di e en me als esul ing in en i onmen al
con amina ion, which dis u bs he na u al mic obial communi ies o hese si es5. Howe e , i is known ha se -
e al o ganisms ha e acqui ed di e se me al esis ance mechanisms such as: (i) change o he me al edox s a e;
(ii) me al cell impe meabili y; (iii) me als p ecipi a ion o liga ion o he cell wall, (i ) up ake and in acellula
chela ion, ( ) e lux mechanism enhancing he me al exc e ion, ( i) sec e ion o me aboli es and ( ii) sec e ion
o me al chela ing agen s (o me aboli es) o he en i onmen 6. Addi ionally, when me als en e in o he cells,
hey o en lead o an inc ease in eac i e oxygen species (ROS) p oduc ion. These species a e e y ins able and
highly eac i e, which esul in nucleic acids damage (mu agenic e ec ), p o ein damage (p o ein oxida ion wi h
loss o unc ion) and memb ane ins abili y ha comp omise he memb ane in eg i y (wi h deg ada ion o he
cell memb ane, lipid pe oxida ion, and/o inhibi ion o elec on anspo chain)7. The e o e, o ganisms ha e
de eloped di e en de ence mechanisms o p o ec cells om hese ha m ul e ec s. These mechanisms can
OPEN
Uni e si y o Coimb a, Cen e o Mechanical Enginee ing, Ma e ials and P ocesses, Depa men o Li e Sciences,
Calçada Ma im de F ei as, 3000-456 Coimb a, Po ugal. *email: [email p o ec ed]
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in ol e ac i a ion o enzyma ic an ioxidan s such as supe oxide dismu ases (SODs), ca alase and pe oxidases
and non-enzyma ic an ioxidan s as glu a hione (GSH) and pigmen p oduc ion8. SOD is conside ed as i s line
o de ence agains oxida i e s ess by con e ing O2− in o H2O2 and ca alase o /and pe oxidases comple e he
de oxi ica ion cycle con e ing H2O2 o O2 and H2O9. In bac e ia, SODs a e he mos impo an enzymes ela ed
o he cellula oxida i e s ess comba and a e o en ca ego ized in di e en amilies based on i s me allic co ac o .
The i s amily has h ee SOD enzymes: Fe-SOD (SOD wi h i on as co ac o ), Mn-SOD (wi h manganese) and
he cambialis ic SOD (which can ha e i on o manganese as he me allic co ac o ). A second amily is composed
by CuZn-SOD (wi h coppe and zinc) and is mainly ound on cy osol o euka yo ic o ganisms. The las one is
Ni-SOD (SOD wi h nickel) and was desc ibed on ma ine ac inomyce es (Ac inobac e ia) and cyanobac e ia10–12.
Many epo s desc ibe he oxida i e s ess p omo ed by hea y me als in bac e ia. Fo example, Rhodobac e
capsula us and Och obac um i ici we e desc ibed o show an inc ease in SOD ac i i y when he cells we e
exposed o ellu i e and ch oma e, espec i ely13,14. Mo eo e , a SOD o e exp ession was also obse ed wi h
Rhodobac e sphae oides cells exposed o seleni e15 and wi h P o eus mi abilis exposed o cadmium and lead16.
The p esence o an ioxidan molecules like glu a hione (γ-GluCysGly, educed o m) (GSH) play a signi ican
ole in sca enging ROS in li ing cells. The p incipal eac ion in ol ing GSH is he hyd ogen pe oxide (H2O2)
deg ada ion o wa e (2GSH + H2O2 → GSSG + 2H2O)17. In bac e ial cells, he e a e some s udies ela ing educed
glu a hione le els wi h esis ance o Esche ichia coli cells o oxida i e s ess condi ions, such as hea y me als,
osmo ic s ess and some an ibio ics18,19. Thus, le els o GSH in cells a e used as a biological pa ame e o e alua e
he cellula oxida i e s ess.
Conce ning he bac e ial in e ac ion wi h Ga and In, he e is e y sca ce in o ma ion. Howe e , Ga seems
o en e in o he bac e ial cells by no comple ely known anspo e s. In li e a u e, some wo ks epo s ha
mu an s s ains (wi h E. coli and Pseudomonas ae uginosa s ains) showed a loss o Ga esis ance when genes
ela ed o i on (Fe) mechanisms we e mu a ed20,21. Those wo ks lead o he hypo hesis ha Fe and Ga me abo-
lisms a e ela ed, and ha Fe-me abolism can a ec Ga esis ance mechanisms. Ano he wo k demons a ed
ha Ga quenches side opho es, ou side he cell, which lead o bac e ia s a a ion and me abolic s ess wi h he
absence o Fe inside he cells22. Up o ou knowledge, he e a e no s udies ha un eil he molecula basis o he
esis ance mechanisms o indium.
In his wo k, wo Se a ia on icola s ains isola ed om me al con amina ed en i onmen s we e used. S. on-
icola s ains a e G am-nega i e bac e ia om En e obac e iaceae amily ha can be ound in di e en en i on-
men s, including d inking wa e , soil, sewage, humans and animals23. Bo h s ains esis an o he c i ical me als
Ga and In we e selec ed o s udy he main e ec s o Ga and In on hese esis an s ains and o co ela e he high
esis ance o hese me als wi h he ac i a ion o biological s a egies o he de oxi ica ion o he me al, namely he
po en ial mechanisms behind he oxida i e s ess gene a ed in esponse o he p esence o hese c i ical me als.
Since he li e a u e ela ed o he p esen subjec is e y sca ce, his s udy will help o unde s and he e ec
o hese wo me als in bac e ial cells and how bac e ial s ains a e able o su i e in p esence o c i ical me als.
Resul s
Minimum inhibi o y concen a ion (MIC) and minimum bac e icidal concen a ion (MBC). The
MIC and MBC alues ob ained o bo h me als, in liquid assays, a e shown in Table1. While he MIC alue o In
was 0.75mM o bo h s ains, he MIC alues o Ga we e 1.5mM and 2mM o s ains A3242 and B2A1Ga1,
espec i ely. The MBC o In was only quan i ied o s ain A3242, because compa ed o o he si ua ions, he
MBC alues a e highe han he maximum concen a ion es ed, 2mMGa and 1mM In.
Analysis o p o ein exp ession. The p o ein exp ession p o ile o bo h s ains g own in p esence o me -
als was e alua ed and compa ed wi h a con ol si ua ion (g own in absence o me als). Figu e1 shows ha bo h
s ains, when exposed o In, o e exp essed a p o ein wi h a molecula weigh o app oxima ely 25kDa (p o ein
band ma ked wi h an a ow). These p o ein bands we e cu and iden i ied by mass spec ome y wi h Masco
se e , using he a ailable UniP o da abase wi hin he axonomic gene a Se a ia, as belonging o i on/manga-
nese supe oxide dismu ase amily.
Me al quan i ica ion. G ow h cu es o he s ains in absence and p esence o Ga and In we e pe o med
o show he e ec o me als on bac e ial g ow h and o selec he ime poin s o collec samples wi h cellula
g ow h enough o me al quan i ica ion assays (Fig.2). Indium had a mo e d as ic e ec in he no mal g ow h
o bo h s ains when compa ed o Ga and his e ec was mo e isible o s ain A3242. Based on g ow h cu es,
he cells o me al quan i ica ion analyses we e aken a wo di e en imes, 6h o g ow h, co esponding o he
end o exponen ial g ow h phase and 24h, co esponding o he la e s a iona y g ow h phase.
Table 1. MIC and MBC alues ob ained o he s ains o indium and gallium.
S ain code
Gallium
concen a ion
(mM)
Indium
concen a ion
(mM)
MIC MBC MIC MBC
A3242 1.50 > 2.00 0.75 > 1.00
B2A1Ga1 2.00 > 2.00 0.75 1.00
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Figu e1. SDS-PAGE (12%) o he p o ein p o ile o bac e ia g own wi hou and wi h me als (0.4mMGa and
0.2mM In): (A) M—low molecula weigh p o ein ma ke (NZYTech), 1—A3242 con ol, 2—A3242 wi h Ga,
3—A3242 wi h In; (B) M—NZYColou p o ein ma ke II (NZYTech), 1—B2A1Ga1 con ol, 2—B2A1Ga1 wi h
Ga, 3—B2A1Ga1 wi h In. The o e exp essed p o ein band was ma ked wi h a ow.
Figu e2. G ow h cu es o s ains A3242 (A) and B2A1Ga1 (B) wi h h ee di e en condi ions: con ol
(wi hou me al), 0.2mMGa and 0.1mM In. Da a shown a e he mean alues (± s anda d de ia ions) ob ained
om wo independen expe imen s.
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The In and Ga up ake by he s ains, analysed by ICP-MS, showed ha s ains ha e di e en le els o me al
accumula ion, as shown in Fig.3. Howe e , bo h s ains showed highe accumula ion o me als a 6h han a 24
h o bac e ial g ow h.
The s ain A3242 showed he highes Ga le els in cells wi h app oxima ely 0.64 and 0.30µgGa/mg p o ein a
6h and 24h, espec i ely, compa ing wi h s ain B2A1Ga1 ha accumula ed app oxima ely 0.13 and 0.10µgGa/
mg p o ein a 6h and 24h, espec i ely. In he case o In, s ain A3242 also showed highe accumula ion a 6h
(app oxima ely 31.31µg In/mg) han s ain B2A1Ga1 (19.53µg In/mg p o ein). A 24h o bac e ial g ow h,
s ains A3242 and B2A1Ga1 only accumula ed 16.40 and 3.35µg In/mg p o ein, espec i ely. Resul s sugges
ha me als a e pumping ou om he cells a he la e s a iona y phase. Quan i ica ion o Ga in he g ow h
medium o he bes me al accumula o , s ain A3232 showed alues o 17,534.3 ± 253ppm, 15,803.8 ± 8.6ppm
and 17,452 ± 35.9ppm a 0h, 6h and 24h, espec i ely. The In alues de e mined in he g ow h medium o he
s ain we e 7961.8 ± 65ppm, 6395.1 ± 17.2ppm and 6947 ± 56.1ppm, espec i ely. Thus, he inc ease o me als
in he g ow h medium a 24h con i med he elease o Ga and In om cells.
Cellula me abolic ac i i y. The cellula me abolic ac i i y was s udied in bo h s ains using he MTT
assay and he esul s a e shown in Fig.4. S ain A3242 e ealed a signi ican dec ease o he cellula me abolic
ac i i y in he p esence o In, showing ac i i y alues o 5.6 and 4.2 old lowe han he con ol, o samples aken
a 6h and 24h o incuba ion, espec i ely. In he case o s ain B2A1Ga1, Ga o In did no a ec signi ican ly i s
me abolic ac i i y.
ROS quan i ica ion. The oxida i e s ess induced by Ga and In was s udied h ough he quan i ica ion o
he in acellula ROS concen a ion in cells exposed o hose me als and compa ed wi h con ol (g ow h wi hou
me als).
In Fig.5A,B, i is possible o obse e ha bo h s ains exhibi ed a signi ican ly highe p oduc ion o ROS in
he p esence o In han in he con ol si ua ion, bu did no show mo e p oduc ion o ROS wi h Ga. The s ain
A3242 showed he highes alues o a io wi h 3.1, 20.3 and 28.3 old highe o 0.1mM, 0.2mM and 0.4mM
o In, espec i ely. S ain B2A1Ga1 showed a io alues o 1.4 and 5.9 old highe han con ol o 0.2mM and
0.4mM o In, espec i ely.
Reduced glu a hione quan i ica ion. Reduced glu a hione was also quan i ied o con i m he po en ial
e ec o Ga and In in he oxida i e s ess induc ion. Reduced glu a hione is conside ed an an ioxidan hiol and
Figu e3. Accumula ion o gallium (A) and indium (B) by s ains A3242 and B2A1Ga1. Da a shown a e he
mean alues (± s anda d de ia ions) ob ained om wo independen expe imen s. ***Signi ican ly di e en ,
p < 0.001.
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lowe concen a ions o educed glu a hione (in ela ion wi h he o al in acellula glu a hione) mean highe
oxida i e s ess.
The Fig.6 shows ha bo h s ains wi h In showed a signi ican ly lowe a io GSH/ o al glu a hione han
he con ol wi h a dec ease o 3.2 and 2.8 old o A3242 and B2A1Ga1, espec i ely). In cells exposu e o Ga,
his a io o bo h s ains was also signi ican ly lowe han in con ol. Howe e , he dec ease o a io GSH/ o al
glu a hione was jus 1.3 and 1.2 old o he con ol o A3242 and B2A1Ga1, espec i ely.
SOD ac i i y in solu ion. The analysis o SOD ac i i y was pe o med o bo h s ains since hey showed
he o e exp ession o a SOD enzyme in he p esence o In ( isible by SDS-PAGE). Figu e7 shows ha bo h
s ains exhibi ed signi ican ly highe SOD ac i i y in he samples wi h In han in he samples wi h Ga o wi hou
me al. Indium exposu e induced an inc ease o app oxima ely wo old o SOD ac i i y compa ed o he con ol
si ua ion wi hou me al.
SOD ac i i y s aining. Soluble p o ein samples o bo h s ains, ob ained om he g ow h in he p esence
and absence o In, we e un in NBT-PAGE elec opho esis. A sample om Esche ichia coli BL21 was used as a
e e ence. To ha e a co ec iden i ica ion o he SOD p esen in he samples, di e en ea men s o he gels we e
pe o med. The e o e, gels we e incuba ed wi h wo di e en inhibi o solu ions: KCN and H2O2.
In absence o inhibi o s (Fig.8A), p o ein samples o bo h s ains om g ow hs in he p esence o In showed
an addi ional SOD band compa ed o he con ol. When incuba ed wi h 10mM H2O2 (Fig.8B), an inhibi o o
Fe-SOD and Cu/Zn-SOD, he lowe band disappea ed in In samples and in he e e ence s ain (E. coli BL21).
Howe e , wi h 10mM KCN (Fig.8C), an inhibi o o Cu/Zn-SOD, he SOD bands we e simila o he con ol
gel (enzyma ic eac ion wi hou inhibi o ), being isible he addi ional lowe SOD band in samples o In. These
esul s sugges ha Se a ia s ains do no ha e Cu/Zn-SOD and ha he induced SOD by In is a Fe-SOD.
Discussion
This wo k is ocused on wo Se a ia s ains, named A3242 and B2A1Ga1, isola ed om he me al-con ami-
na ed mines o U gei iça and Panasquei a. U gei iça en i onmen is na u ally con amina ed wi h u anium24 and
Panasquei a wi h ungs en bu o he me als a e also p esen in small o ace quan i ies25. Despi e hese si es a e
no epo ed as gallium o indium con amina ed, bo h isola es showed ex ao dina y esis ance o bo h c i ical
Figu e4. MTT assays o s ains A3242 (A) and B2A1Ga1 (B). Da a shown a e he mean alues (± s anda d
de ia ions) ob ained om h ee independen expe imen s. ***Signi ican ly di e en om he alue o Con ol
(wi hou me al), p < 0.001, espec i ely. Rela i e Op ical Densi y Uni s (RODU) means he a io be ween he
OD550 (abso bance a 550nm) o he sample ( es ) and he OD550 o he con ol expe imen .

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me als, exhibi ing high MIC alues (0.75mM o In and 1.5mM o 2mM o Ga) while a la ge numbe o isola es,
including o he es ed Se a ia s ains, showed MICs o 1mM o Ga and 0.25mM o In as he highes alues.
The na u al p esence o me als in hese en i onmen s may p essu e he au och honous bac e ial communi ies,
selec ing he mos esis an mic oo ganisms o me als in gene al.
The e a e e y ew wo ks explo ing Bac e ia-Ga/In in e ac ions, he e o e, his wo k aimed o unde s and
he biological mechanisms behind oxida i e s ess gene a ed in esponse o exposu e o c i ical me als and i s
con ol by he cells. In his sense, hese wo e y esis an s ains we e s udied conce ning he di e en ial p o ein
exp ession (unde o o e exp ession) when g own wi h hose c i ical me als, isible on SDS-PAGE gels. Only
In induced de ec able o e exp ession o p o eins, iden i ied as SOD enzymes. These enzymes a e usually ela ed
o he con ol o cellula oxida i e s ess9.
The SOD ac i i y de ec ed in he soluble p o ein ac ions was used o iden i y he addi ional SOD induced by
In. I is known ha KCN does no inhibi bo h Fe-SOD and Mn-SOD (inhibi s CuZn-SOD) and H2O2 does no
inhibi Mn-SOD bu inhibi s Fe-SOD and CuZn-SOD26. In his wo k, he inhibi o y eac ions we e compa ed
enabling he iden i ica ion o he me allic co ac o p esen in he addi ional SOD enzyme. I was inhibi ed by
H2O2 bu no by KCN concluding ha his enzyme was a Fe-SOD. Conside ing all he esul s obse ed wi h
bo h SOD ac i i y assays, i is possible o conclude ha bo h s ains ha e highe SOD ac i i ies when exposed o
In. P e ious wo k, ela ing he genome o Se a ia sp. LCN16 s ain wi h he high ole ance o oxida i e s ess
p edic ed h ee genes, encoding h ee SOD-enzymes: wo Fe/Mn-SOD (sod2) and one Cu/Zn-SOD (sod1)27. Up
o ou knowledge, cu en ly he e is no wo k ela ing he induc ion o SODs in Se a ia s ains wi h he p esence
o Ga and In. Ne e heless, o some s ains, i was al eady desc ibed an o e exp ession o one speci ic SOD in
he p esence o hea y me als, such as o e exp ession o Mn-SOD wi h cadmium and lead in P o eus mi abilis,
and o e exp ession o Fe-SOD wi h ch oma e in Rals onia me allidu ans16,28. A ecen s udy epo ed a g adual
and signi ican inc ease o he SOD ac i i y in p esence o an imony concen a ions up o 10mM in a s ain o
Se a ia ma cescens isola ed om oo s o he Hedysa um pallidum plan 29.
Figu e5. Ra io alues o he ela i e luo escence uni s (RFU o he es assay/RFU o he con ol assay)
ob ained o s ains A3242 (A) and B2A1Ga1 (B). Da a shown a e he mean alues (± s anda d de ia ions)
ob ained om h ee independen expe imen s. ***Signi ican ly di e en om he alue o con ol (wi hou
me al), p < 0.001.
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I is well epo ed ha me als a e o en esponsible o oxida i e s ess in he cell (one cellula consequence
om me al s ess), which can a ec he bac e ial g ow h and e en esul in cell dea h30. Oxida i e s ess can be
analysed using di e en app oaches, such as ROS quan i ica ion using p obes, p o ein damage quan i ica ion
(p o ein ca bonyl con en ), lipid pe oxida ion e alua ion (malondialdehyde con en ), DNA damage isualiza ion,
measu emen o non-enzyma ic an ioxidan s (e.g. educed glu a hione) and assessmen o enzyma ic an ioxidan s
(e.g. supe oxide dismu ase, ca alase ac i i ies)31. In his wo k, he ROS le els quan i ied o bo h selec ed s ains
e ealed ha , in gene al, he s ains showed a high ROS concen a ion when incuba ed wi h high concen a ions
o In. This is he i s s udy o clea ly show he e ec o In sal s in he inc ease o ROS p oduc ion in bac e ial
cells. P e ious wo ks only showed he consequence o Indium Tin Oxide (ITO) nanopa icles exposu e in he
inc ease o he in acellula ROS le els on human cells32,33. The s ains did no show a high p oduc ion o ROS
when exposed o high Ga concen a ions, sugges ing ha Ga (a es ed concen a ions) did no induce oxida i e
s ess in hose bac e ia. These esul s a e acco ding o he analysis o he SDS-PAGE p o ile. In he p esence o
In, bo h bac e ia showed high oxida i e s ess, which esul ed in high SOD ac i i y o de oxi y he cellula ROS.
The oxida i e s ess induced by he a ge c i ical me als was also con i med by quan i ica ion o he educed
glu a hione le els. This an ioxidan exis s in he educed o m in low quan i ies inside he cells when he cells
a e unde oxida i e s ess condi ions19. Bo h s ains showed lowe a ios ( educed glu a hione/ o al glu a hione)
when subjec ed o he c i ical me als. The a io dec ease was especially ele an in he p esence o In. This las
esul is in ag eemen wi h he ROS le els de ec ed in cells, con i ming ha in S. on icola, In induces high
oxida i e s ess.
The e ec o Ga and In on cellula me abolic ac i i y was assessed by MTT assays. The dec ease o ac i i y
in cells is conside ed a good indica o o cell edox ac i i y34. The s ains we e a ec ed by he exposi ion o bo h
c i ical me als, bu indium exhibi ed he mos d as ic oxic e ec . Analysing he esul s o cellula iabili y, he
s ains, despi e belonging o he same species, did no show an iden ical p o ile when exposed o he c i ical
me als.
The wo Se a ia s ains A3242 and B2A1Ga1 come om di e en en i onmen s and his ac can jus i y he
no able di e ence in he cellula iabili y o hese s ains when g own wi h In. The di e ences be ween s ains
Figu e6. Ra io alues be ween he educed glu a hione and he o al in acellula glu a hione o s ains A3242
(A) and B2A1Ga1 (B). Da a shown a e he mean alues (± s anda d de ia ions) ob ained om wo independen
expe imen s. ***Signi ican ly di e en om he alue o con ol (wi hou me al), p < 0.001.
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could also be ela ed o di e en me al up akes. The s ain able o accumula e mo e me al showed he s ong-
es dec ease in me abolic ac i i y. I is known ha high amoun s o me al inside he cells migh esul in many
oxic e ec s in cells, esul ing in lowe cellula iabili y13. S ains showed he end o pump me al ou o cells
a he la e s a iona y phase (24h o incuba ion) o g ow h. Me al e lux mechanisms (p esen o inducible) in
bac e ia dec ease he cellula damage esul ing om me al accumula ion and a e o en esponsible o bac e ial
Figu e7. SOD ac i i y (U/mg p o ein) o s ains A3242 (A) and B2A1Ga1 (B), in absence and p esence o
he c i ical me als, Da a shown a e he mean alues (± s anda d de ia ions) ob ained om wo independen
expe imen s. *,**Signi ican ly di e en om he alue o con ol (wi hou me al), p < 0.05 and p < 0.01,
espec i ely.
Figu e8. Ac i i y s aining o he p o ein samples elec opho esed on h ee independen 10% nondena u ed
polyac ylamide gels: (A) enzyma ic eac ion in absence o inhibi o s; (B) enzyme incuba ed wi h 5mM H2O2;
(C) enzyme incuba ed wi h 5mM KCN. A ows indica ed he addi ional SOD band.O iginal gels shown in
Fig.S1.
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me al esis ance30,35,36. The e is no desc ip ion o mechanisms o pump-ou In and Ga om he cells. Howe e ,
conside ing he e ec o bo h me als in he cellula iabili y, i is possible ha bac e ia pump-ou me al in he
s a iona y phase o limi he cellula damage (DNA, p o eins, lipids). Fo ins ance, s udies wi h E. coli and Bacil-
lus sub ilis showed ha genes coding o pu a i e manganese e lux pumps a e up egula ed when he bac e ia
a e exposed o manganese37,38.
In conclusion, he c i ical me als ocused on his wo k, exhibi ed di e en impac in wo S. on icola s ains.
Bo h s ains when exposed o Ga jus showed a signi ican ly dec ease o he a io GSH/ o al glu a hione, while
hey exhibi ed an inc ease o oxida i e s ess when exposed o In, wi h ac i a ion o biological mechanisms o
con ol ha s ess, such as educed glu a hione and SOD enzymes. These s ains we e e en able o ac i a e an
addi ional SOD enzyme (Fe-SOD) as a esponse o he oxici y induced by In. Unde s anding he s a egies o
me al esis an bac e ia o cope wi h c i ical me als is impo an o enable and p omo e he use o bac e ia in
inno a i e and sus ainable p ocesses o me al eco e y.
Me hods
Bac e ial s ains, media and g ow h condi ions. S. on icola A3242 (isola ed om U gei iça mine)
and S. on icola B2A1Ga1 (isola ed om Panasquei a mine) we e g own in Reasone ’s 2A b o h medium (R2Ab),
con aining pe li e : 0.5g o yeas ex ac , 0.5g o p o eose pep one, 0.5g o casein, 0.5g o glucose, 0.5g o solu-
ble s a ch, 0.3g o K2HPO4, 0.024g o MgSO4 and 0.3g o sodium py u a e. Bac e ial g ow h was e alua ed a e
incuba ion a 30 ºC, measu ing he op ical densi y a 600nm (OD600). Me al s ock solu ions we e p epa ed in a
concen a ion o 0.5M indium(III) chlo ide (InCl3) (Ac os O ganics) and 0.2M gallium(III) ni a e (GaN3O9)
(Al a Aesa ) and we e s e ilized by il a ion.
Minimum inhibi o y concen a ion (MIC) and minimum bac e icidal concen a ion (MBC)
assays. The MIC o Ga and In we e e alua ed using he s anda d b o h mic odilu ion me hod in R2Ab39.
Each assay was epea ed in iplica e.
Addi ionally, he assays wi h me al concen a ions equal and abo e he MIC alues ob ained we e used o
de e mina e he MBC. The e o e, bac e ial suspensions om he wells wi h Ga o In concen a ions ha did
no show bac e ial g ow h we e pla ed on o R2A solid medium. Bac e ial g ow hs we e analysed a e 48h o
incuba ion a 30 ºC.
Analysis o p o ein exp ession. The bac e ial esis ance mechanisms o me als migh be ela ed o di -
e en ial exp ession (unde o o e exp ession) o speci ic p o eins. The e o e, he p o ein exp ession p o iles o
cells g own in he absence (con ol) o p esence o c i ical me als (0.1mM In o 0.2mMGa) we e e alua ed
by sodium dodecyl sulpha e–polyac ylamide gel elec opho esis (SDS-PAGE). Fi s ly, he cells om 3days o
g ow h we e cen i uged and washed wice wi h Phospha e Bu e Saline solu ion (PBS—8g/l NaCl, 0.2g/l KCl,
1.44g/l Na2HPO4, 0.24g/l KH2PO4, pH 7.4). The pelle was esuspended in 300µl o PBS and dis up ed by ou
cycles o 30s pulse sonica ion (Sonics & Ma e ials Inc. Danbu y, Connec icu U.S.A.) a 60 A. The samples we e
cen i uged a 13,000 pm ( o a ion pe minu e) o 15min and he p o ein con en in each ac ion was quan i-
ied by B ad o d me hod40. The sea ch o a di e en ial p o ein exp ession p o ile was e alua ed by 0.1% sodium
dodecyl sul a e (SDS)—12% polyac ylamide gel elec opho esis (PAGE), wi h a Coomassie Blue s aining. The
p o ein bands o e exp essed in p esence o he me als we e iden i ied by pep ide mass spec ome y wi h Mas-
co se e (Maldi-TOF, IPATIMUP, Po o).
Me al quan i ica ion. S ains we e g own in R2Ab a 30 ºC wi h 140 pm spiked wi h 0.2mMGa and
0.1mM In. Samples o bac e ial g ow h we e collec ed a 6 hand 24h.
The samples we e cen i uged h ee imes a 4000 pm o 20min a 4 ºC, he cellula pelle s we e washed
wice wi h cold PBS solu ion and he inal bac e ial pelle s we e lysed wi h an acid ea men (5% HNO3), hea ed
a 50 ºC o 1h and hen cen i uged a 13,000 pm o 10min. The in acellula supe na an s and he medium
om bac e ial g ow h a a speci ic ime o sampling (0h, 6h and 24h) we e dilu ed o he me al quan i ica ion
by Induc i ely Coupled Plasma Mass Spec ome y (ICP-MS)41. Pelle s we e neu alized wi h NaOH 0.5M and
hen used o quan i y he o al p o ein by B ad o d me hod40.
Tes o cellula me abolic ac i i y. The cellula me abolic ac i i y was e alua ed using he 3-(4,5-dime h-
yl hiazol-2-yl)-2,5-diphenyl e azolium b omide assay (MTT assay), acco ding o he p o ocol o Wang and
colabo a o s42 modi ied as desc ibed. B ie ly, 1ml o cellula g ow h, aken a he incuba ion imes p e iously
e e ed, we e cen i uged, washed wice wi h R2Ab and he pelle s we e esuspended in 1ml o medium. The
cell suspensions we e dilu ed wi h R2Ab o an OD600 o 0.2 and mixed 10:1 wi h MTT s ock solu ion (5g/ml).
The mix u es we e incuba ed wi h he cap ube open a 30 ºC o 1h. A e he incuba ion ime, he mix u es
we e cen i uged a 10,000g o 2min and he pelle s dissol ed in 2.5ml o dime hyl sul oxide (DMSO). The
samples we e incuba ed 1h a oom empe a u e be o e quan i ied spec opho ome ically a 550nm.
ROS quan i ica ion. ROS concen a ion in he cells was quan i ied using he 2′,7′-dichlo odihyd o luo es-
cein diace a e (H2DCFDA) me hod43. The p o ocol ollowed was based on he used me hod in a p e ious wo k14
and was op imized o he cu en assays wi h Ga and In. S ains g own in R2Ab, we e exposed o di e en me al
concen a ions when g ow h eached OD600 o 0.2–0.3, and 2h la e , exposed o 25μM o H2DCFDA o 1h. The
cells we e cen i uged, washed wice wi h PBS and he pelle s we e esuspended in 1ml PBS. The luo escence
(λem = 527nm and λex = 495nm) and OD600 we e ead hou ly du ing 5h. ROS a e de e mined as he Rela i e Flu-