Academic Edi o s: Juan M. Gonzalez
and Alba Cuecas
Recei ed: 23 Decembe 2024
Re ised: 13 Janua y 2025
Accep ed: 16 Janua y 2025
Published: 21 Janua y 2025
Ci a ion: Pe ez-Cas iñei a, J.R.;
Á ila-Oli a, F.J.; Se ano, A.
Enginee ing Ino ganic Py ophospha e
Me abolism as a S a egy o Gene a e
a Fluo ide-Resis an Saccha omyces
ce e isiae S ain. Mic oo ganisms 2025,
13, 226. h ps://doi.o g/10.3390/
mic oo ganisms13020226
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Enginee ing Ino ganic Py ophospha e Me abolism as a S a egy
o Gene a e a Fluo ide-Resis an Saccha omyces ce e isiae S ain
José R. Pe ez-Cas iñei a 1,* , F ancisco J. Á ila-Oli a 2and Au elio Se ano 1
1Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa-CSIC, A . Amé ico Vespucio 49,
41092 Se illa, Spain; [email p o ec ed]
2Apli e, 41703 Dos He manas, Spain; [email p o ec ed]
*Co espondence: j [email p o ec ed]
Abs ac : Fluo ine accoun s o 0.3 g/kg o he Ea h’s c us , being widely dis ibu ed in
he en i onmen as luo ide. The oxic e ec s o his anion in humans and o he o ganisms
ha e been known o a long ime. Fluo ide has been epo ed o al e se e al cellula
p ocesses al hough he mechanisms in ol ed a e la gely unknown. Ino ganic py ophos-
pha ases (PPases) a e ubiqui ous enzymes ha hyd olyze ino ganic py ophospha e (PPi), a
me aboli e gene a ed om ATP. In Saccha omyces ce e isiae, he enzyme esponsible o PPi
hyd olysis in he cy osol (IPP1) is s ongly inhibi ed by luo ide
in i o
. The essen iali y
o IPP1 o g ow h has been p e iously demons a ed using YPC3, a yeas mu an wi h
condi ional exp ession o he co esponding gene. He e, YPC3 was used o gene a e cells
ha ole a e high concen a ions o luo ide by (a) he o e exp ession o IPP1 o i s human
o holog, o (b) he subs i u ion o IPP1 by he luo ide-insensi i e PPase om S ep ococcus
mu ans. The esul s ob ained sugges ha main aining app op ia e le els o PPase ac i i y
in he cy osol is essen ial o he adap a ion o S. ce e isiae o high luo ide concen a ions.
The inc ease in luo ide ole ance allows YPC3 cells ans o med wi h sui able plasmids o
be selec ed on ich non-selec i e medium supplemen ed wi h his anion.
Keywo ds: luo ide esis ance; ino ganic py ophospha ase; o e exp ession; Saccha omyces
ce e isiae;S ep ococcus mu ans
1. In oduc ion
Fluo ide, he smalles o he halide anions, has he highes elec onega i i y o all
he elemen s o he Pe iodic Table. I is e y abundan in he Ea h’s c us (an a e age o
0.32 g/kg), being ound in soils, wa e and he ai . Fluo ide concen a ions ange om
25
µ
M o 100 mM in wa e esou ces, and i may be he mos abundan anion in ce ain
g oundwa e s [
1
,
2
]. Consump ion o wa e wi h a luo ide con en close o 0.2 mM can
p oduce den al luo osis in child en, while skele al luo osis, a mo e se e e disease, may
esul om egula ly d inking wa e wi h highe le els o luo ide (0.2 o 0.8 mM). Skele al
luo osis causes back pain and igidi y as well as neu ological diso de s, being endemic in
a leas 25 coun ies including India, Mexico, Kenya and Tanzania. The numbe o people
su e ing om his disease is es ima ed o be in he ange o ens o millions [
3
]. Fluo ide
also exe s e ec s on o he o ganisms such as bac e ia and ungi. The an imic obial ac i i y
o his anion has been s udied in ecen decades, mainly on bac e ia causing den al ca ies,
and i has been epo ed o be oxic o yeas and o he ungi, some o hem pa hogenic [4].
Due o i s ubiqui y in he en i onmen and i s oxic e ec s, many o ganisms ha e
de eloped s a egies o luo ide esis ance, al hough li le is known abou hese s a egies
Mic oo ganisms 2025,13, 226 h ps://doi.o g/10.3390/mic oo ganisms13020226
Mic oo ganisms 2025,13, 226 2 o 18
and he biochemical pa hways in ol ed. Fluo ide- esponsi e iboswi ches ha egula e
he exp ession o genes in esponse o his anion ha e been iden i ied in bac e ia and
a chaea, luo ide expo e s being he mos common p o eins ound in ope ons wi h hese
iboswi ches [
5
,
6
]. Two di e en luo ide expo p o eins ha e been iden i ied in p oka y-
o es: (a) CLC
F
s, luo ide/p o on an ipo e s, which ha ness he p o on g adien o expel
luo ide om he cy oplasm, and (b) Fluc p o eins ( luo ide channels), passi e channels ha
d i e luo ide ex usion down i s elec ochemical g adien ac oss he plasma memb ane.
Among he euka yo es, ungi (including yeas ), plan s, and some ocean-dwelling animals
possess luo ide expo e s ha belong o a hi d p o ein amily known as FEX, s uc u ally
ela ed o he Flucs [
6
,
7
]. Besides ex usion sys ems, al e na i e ac o s ha e been epo ed
o in luence luo ide ole ance [6,8].
Fluo ide is known o be an inhibi o o ce ain majo in acellula p o eins, and i s
e ec s a e belie ed o be exe ed by cha ge in e ac ion wi h ca ions such as Mg
2+
and/o
Ca
2+
. In he case o enolase, s uc u al da a sugges ha he inhibi ion is due o he
o ma ion o a magnesium- luo ide–phospha e complex [
9
]. Fluo ide also has he capaci y
o o m complexes wi h aluminum and be yllium ca ions ha may ac as phospha e
emula o s, hus inhibi ing enzymes in ol ed in he ans e o phospho yl g oups [
10
].
This la ge class o enzymes include he so-called ino ganic py ophospha ases (PPases,
EC 3.6.1.1), which hyd olyze ino ganic py ophospha e (PPi) [
11
]. PPi is a by-p oduc o
many anabolic eac ions, and i s e icien emo al allows hei shi owa d biosyn hesis
acco ding o he law o mass ac ion [
12
]; as a consequence, accumula ion o PPi can
collapse anabolism in cellula compa men s like cy osol o mi ochond ia. Two main
classes o s uc u ally di e en PPases ha e been iden i ied o da e: (a) soluble PPases
(sPPases), ubiqui ous p o eins ha hyd olyze PPi, eleasing he chemical ene gy o he
phosphoanhyd ide bond as hea , and (b) ion (H
+
and/o Na
+
)- ansloca ing ino ganic
py ophospha ases (mPPases), in eg al memb ane p o eins ha couple PPi hyd olysis o
p o on and/o sodium pumping ac oss biological memb anes [
13
,
14
]. Among he sPPases,
wo majo non-homologous amilies, known as I and II, ha e been cha ac e ized [
15
–
17
]
(Figu es S1 and S2). Family I sPPases occu in all ypes o o ganisms, bo h p oka yo ic and
euka yo ic [
18
]. In his amily o sPPases, PPi binding o he ac i e si e is a complex p ocess
ha depends on di alen ca ion co ac o s, Mg
2+
being he mos e icien . The e olu iona ily
un ela ed amily II sPPases, which belong o he DHH-DHHA2 phosphoes e ase p o eins,
a e ac i a ed by o he hea y-me al ca ions such as Mn
2+
o Co
2+
(al hough Mg
2+
ac s
as a co- ac o ). Family II sPPases include hose om S ep ococcus mu ans (a bac e ium
ha con ibu es o oo h decay in he human o al ca i y), Bacillus sub ilis and o he G am-
posi i e bac e ia (Fi micu es), as well as some o he lineages o bac e ia (Chlo o lexi,
Spi ochae es, The mo ogae) and a chaea (Eu ya chaeo a). Family I sPPases a e s ongly
inhibi ed by 0.5 mM luo ide
in i o
, whe eas much highe concen a ions o his anion
a e needed o inhibi amily II sPPases and mPPases [19–21].
Saccha omyces ce e isiae has wo genes encoding sPPases, bo h belonging o amily I:
IPP1 and IPP2 (also known as PPA2) (Figu e S1). IPP1 is conside ed an essen ial nucleo-
cy oplasmic p o ein while IPP2 is epo edly loca ed in he mi ochond ial lumen, whe e
i plays a ole in espi a o y me abolism [
22
–
24
]. P e ious wo k om ou g oup in bud-
ding yeas showed ha cells de oid o IPP1 unde go di e en a es depending on hei
ene gy me abolism; hus, e men ing cul u es show massi e cell dea h by au ophagy, while
espi ing cul u es do no die bu unde go g ow h a es in he S-phase o he cell cycle [
25
].
In human cells, he e a e wo amily I sPPase pa alogs—PPA1 and PPA2—acco ding o
he UNIPROT da abase. These p o eins a e loca ed a he cy oplasm and mi ochond ia,
espec i ely, a si ua ion simila o ha o S. ce e isiae.
Mic oo ganisms 2025,13, 226 3 o 18
The essen iali y o IPP1 o yeas g ow h, along wi h p e ious epo s showing he
high sensi i i y o amily I sPPases o luo ide
in i o
, p omp ed us o check whe he his
in e ac ion could ha e implica ions
in i o
. The yeas mu an s ain YPC3, p e iously
gene a ed in ou labo a o y [
26
], was used o accomplish his ask. YPC3 cells ha e hei
IPP1 gene (SGD sys ema ic name, YBR011C) unde he con ol o he yeas galac okinase
gene (GAL1) p omo e ; consequen ly, hey only exp ess hei nucleocy osolic sPPase, when
g own on galac ose, being unable o g ow on o he ca bon sou ces. YPC3 cells egain he
capaci y o g ow on glucose ( e men a i e condi ions) o glyce ol ( espi a o y condi ions)
by ans o ma ion wi h plasmids bea ing PPase genes unde he con ol o cons i u i e
p omo e s [
24
–
28
]. In p ac ical e ms, YPC3 allows he subs i u ion o IPP1 by di e en
PPi-hyd olyzing enzymes, hence he compa ison be ween di e en ypes o PPases wi hin
he same cellula con ex .
He e, we show ha he esis ance o S. ce e isiae cells o luo ide can be inc eased by
o e exp essing IPP1 o i s human amily I o holog, PPA1. Fu he mo e, yeas cells in which
IPP1 is unc ionally subs i u ed by ela i ely low le els o he luo ide- esis an amily II
sPPase om S. mu ans may also esul in cells wi h highe ole ance o his anion. These
esul s sugges a majo ole o IPP1 in he adap a ion o S. ce e isiae o high concen a ions
o luo ide and shed ligh on he cy o oxic e ec s exe ed by his anion a he molecula
le el. Possible implica ions o o he euka yo ic and p oka yo ic cells a e discussed.
2. Ma e ials and Me hods
2.1. Bac e ia and Yeas S ains
Esche ichia coli DH5
α
s ain (supE44 lacU169 (Ø80 lacZ M15) hsdR17 ecA1 endA1
gi A46 hi-1 el A1) [
29
] was used o cloning pu poses. S. ce e isiae haploid s ain W303-1A
(MATa, ade2-1 can1-100 his3-11,15 leu2-3,112 p1-1, u a3-1) [
30
] was used bo h as a wild
ype and as a pa en al s ain o gene a e a YPC3 mu an (W303-1A ipp1
UAS
-ipp1
TATA
::HIS3-
GAL1
UAS
-GAL1
TATA
-IPP1) by he single-s ep ansplacemen p ocedu e, as p e iously
desc ibed [26].
2.2. Plasmids Cons uc ion and Yeas T ans o ma ion
Plasmids used o yeas ans o ma ion, lis ed in Table 1, we e, in mos cases, de i a-
i es o he high-copy pRS699b plasmid [
26
,
31
] o he low-copy pRS416 plasmid [
32
]. The
coding sequence o he IPP1 gene was ampli ied by PCR along wi h a 400 bp p omo e
egion ups eam he s a codon, as p e iously desc ibed [
26
]. The la e has been shown o
con ain all he cis-ac ing elemen s essen ial o e icien IPP1 ansc ip ion [
33
]. A i icial
EcoRI and SpeI es ic ion si es we e in oduced a he 5
′
and 3
′
ends, espec i ely, in o de
o di ec ionally inse he esul ing DNA agmen in o he pRS699b plasmid. This yielded
plasmid pIPP1-699, in which IPP1 is inse ed be ween i s own p omo e and he PMA1 gene
e mina o egion. The coding sequence o he S. mu ans amily II sPPase (SPP2; gene locus
DQM59_RS02590) was ampli ied om genomic DNA by PCR wi h a i icial EcoRV and
SpeI si es a i s 5
′
and 3
′
-ends, espec i ely, and in oduced in pIPP1-699 using an EcoRV
na u al es ic ion si e loca ed jus ups eam he s a codon o IPP1 and he unique SpeI
si e, hus yielding plasmid pSPP2-699. The exp ession casse es con aining he 400 bp long
IPP1 p omo e egion, he coding sequences o IPP1 o SPP2, and he PMA1 e mina o
egion we e clea ed ou o plasmids pIPP1-699 and pSPP2-699 wi h es ic ion enzymes
EcoRI and HindIII and liga ed o plasmid pRS416, diges ed wi h he same enzymes. This
yielded plasmids pIPP1-416 and pSPP2-416. A simila s a egy was ollowed o ob ain
plasmid piGMVP-416 om a p e iously published plasmid encoding he ion- ansloca ing
memb ane PPase (mPPase) o he me hanogenic a chaeon Me hanosa cina mazei (MVP) [
28
]
(Figu e S3), which is unc ionally and s uc u ally e y di e en o he sPPases used in his
Mic oo ganisms 2025,13, 226 4 o 18
wo k. High-copy plasmid pPPA1-426, bea ing he cDNA coding o he cy osolic sPPase
PPA1 om Homo sapiens (PPA1; HGNC, locus NC_000010.11) inse ed in he high-copy
plasmid pRS426 [
34
] unde he con ol o he yeas glyce aldehyde-3-phospha e dehyd o-
genase (GPD) p omo e , was gene ously dona ed by D . Agus ín He nández (Be e nos ic
S. L., Noáin, Na a a, Spain). Finally, plasmid pIPPGFP-699 had been p e iously ob ained
by ou g oup [24].
S. ce e isiae mu an s ain YPC3 was ans o med wi h he plasmids desc ibed abo e
by using he me hod o Schies l and Gie z [
35
]. Cells we e ini ially g own a 30
◦
C in
ich medium con aining galac ose [YPGal: 1% (w/ ) yeas ex ac , 2% (w/ ) pep one, 2%
(w/ ) galac ose] and ans o man s we e selec ed by g owing cells on 2% (w/ ) aga pla es
made in syn he ic medium [0.17% yeas ni ogen base wi hou amino acids and ammonium
sul a e, 2% galac ose, 50 mM MES-TRIS pH 6, and a mix u e o nucleo ides and amino
acids, desc ibed elsewhe e [36], de oid o his idine and u acil].
2.3. Pheno ype Complemen a ion Tes s
Fo complemen a ion s udies, 2 mL o YPD [1% (w/ ) yeas ex ac , 2% (w/ ) pep one,
2% (w/ ) glucose] was inocula ed wi h ans o med cells om he pla es and cul u ed
o e nigh a 30
◦
C wi h agi a ion (200 .p.m. in a In o s O bi on o bi al shake ) un il he
s a iona y phase was eached. An amoun o 20
µ
L o hese cul u es was used o inocula e
2 mL o YPD and g own again o e nigh unde he same condi ions. This ea men was
necessa y o dec ease he py ophospha ase ac i i y associa ed wi h IPP1 in YPC3 cells. Fo
d op es s, en- old se ial dilu ions o he inal cul u es we e made in s e ile wa e , and
2.5
µ
L d ops o each dilu ion we e spo ed on o 2% (w/ ) aga pla es made in YPD o
YPGly (made as YPD wi h 3% (w/ ) glyce ol subs i u ing o glucose). Cul u e media we e
bu e ed by he addi ion o 50 mM MES (adjus ed o he speci ied pH alues wi h TRIS)
and supplemen ed wi h NaF a he indica ed concen a ions. Pla es we e ypically g own
a 30 ◦C o 3–4 days.
2.4. G ow h Cu es in Liquid Medium
Cells we e cul u ed as desc ibed abo e, excep ha liquid YPD medium adjus ed o
pH 5.0 wi h 50 mM MES-TRIS and supplemen ed wi h 75 mM NaF was used ins ead o
aga pla es. Samples we e collec ed a indica ed imes and hei op ical densi y a 660 nm
(OD660) was measu ed.
2.5. P epa a ions o Soluble P o ein Ex ac s om Yeas
Yeas colonies we e collec ed om a pla e and liquid-g own up o he s a iona y phase
in selec i e medium; hen, 2–4 mL o YPD was inocula ed wi h a 1:100 olume o s a iona y
cul u e. A e o e nigh g ow h a 30
◦
C wi h agi a ion as desc ibed abo e, cells we e
sedimen ed by cen i uga ion in a bench cen i uge (3000
×
g, 5 min), washed ho oughly
wi h wa e , esuspended in 0.2 mL o ice-cold bu e A (25 mM T is-HCl, pH 8, 10% (w/ )
glyce ol, 2 mM DTT, 1 mM EDTA, 1 mM benzamidine, 2 mM
ε
-aminocap oic acid, 1 mM
PMSF), and homogenized by igo ous shaking wi h glass beads. The homogena e was
dilu ed up o 0.5 mL wi h bu e B (10 mM T is-HCl, pH 8, 10% glyce ol, 2 mM DTT, 1 mM
EDTA) and cen i uged in a mic o uge (20,000
×
g, 20 min) a 4
◦
C o emo e beads, deb is,
and mos o he memb ane ac ion. The esul ing supe na an was used as he soluble
p o ein ex ac .
2.6. Enzyma ic Assay, P o ein De e mina ions and Wes e n Blo ing
Py ophospha ase ac i i y was assayed a 30
◦
C in a inal olume o 200 mL in a
medium con aining 50 mM MOPS-TRIS pH 7.2, 1 mM MgCl
2
and 0.5 mM Na
4
PPi in he
p esence and absence o 0.5 mM NaF. The amoun o eleased o hophospha e was de e -
Mic oo ganisms 2025,13, 226 5 o 18
mined spec opho ome ically, as p e iously desc ibed [
37
]. In e e y assay, he amoun o
sample and/o he incuba ion ime (usually 3–5 min) we e adjus ed so ha he abso bance
alues ell wi hin he linea pa o a calib a ion cu e made wi h a comme cial o hophos-
pha e s anda d solu ion. Abso bances we e measu ed e e y 20 s, plo ed e sus ime, and
he ac i i ies we e calcula ed om he slopes o he esul ing s aigh lines. P o ein con-
cen a ion was es ima ed using a Coomassie-blue dye binding-based assay om Bio-Rad
(München, Ge many) acco ding o he manu ac u e ’s ins uc ions using o albumin as a
s anda d. Immunode ec ion by Wes e n blo was pe o med as desc ibed elsewhe e [
38
]
using a comme cial abbi polyclonal an ibody agains S. ce e isiae IPP1 (AP21326F_N,
O iGene EU, He o d, Ge many).
2.7. De e mina ion o In e nal PPi Le els in Yeas Cells
YPC3 cells ans o med wi h he plasmids shown in Table 1we e g own in YPD
bu e ed a pH 5, as desc ibed abo e. The ini ial OD
660
alues o he cul u es we e indi-
idually adjus ed so ha a e 8–9 h, all o hem had a inal OD
660
o a ound 0.5 ( oughly
10
9
cells). Cul u es we e hen spli in o wo aliquo s, and NaF ( inal concen a ion 75 mM)
was added o one o hem. Cells we e g own o wo mo e hou s unde he same condi ions,
a e which hey we e collec ed by cen i uga ion, washed wi h ice-cold deionized wa e
and b oken as desc ibed in Sec ion 2.5, excep ha a 4% ( / ) pe chlo ic acid aqueous
solu ion was used ins ead o bu e A. Beads, deb is, and dena u ed p o eins we e emo ed
by cen i uga ion (20 min, 20,000
×
g, 4
◦
C). PPi concen a ions we e de e mined in he
supe na an s as p e iously desc ibed [39].
Table 1. Plasmids used in his communica ion.
Plasmid Desc ip ion Re e ences
pRS699b 1
S.ce e isiae/E. coli shu le plasmid bea ing he cons i u i e
p omo e and e mina o o yeas gene PMA1 and selec ion
ma ke URA3. I has a 2-mic on (2 m) o igin o eplica ion,
which yields a high numbe o plasmid copies (14–34) pe cell.
[31]
pIPP1-699 1,2 Plasmid de i ed om pRS699b bea ing he p omo e and
coding sequence o gene IPP1 om S. ce e isiae inse ed
ups eam o he PMA1 e mina o .
[40]
pIPP1-416 1
Plasmid de i ed om pRS416 bea ing he p omo e and coding
sequence o gene IPP1 om S. ce e isiae. I s main di e ence
om pIPP1-699 is ha i has a yeas cen ome ic sequence (CEN)
and an au onomously eplica ing sequence (ARS) ha esul s in
a low numbe o plasmid copies (2–5) pe yeas cell.
This s udy
pSPP2-699 1Plasmid de i ed om pIPP1-699 bea ing gene SPP2 om
S. mu ans.This s udy
pSPP2-416 1Plasmid de i ed om pIPP1-416 bea ing gene SPP2 om
S. mu ans.This s udy
pPPA1-426 1
Plasmid de i ed om high-copy plasmid pRS426 ha bea s he
cDNA coding o he cy osolic sPPase om Homo sapiens (PPA1).
Dona ed by
D . He nández
piGMVP-416 1,2
Plasmid de i ed om pIPP1-416 bea ing he sequence ha
codes o he pu a i e N- e minal signal pep ide o yeas Suc2p
ollowed by hose o yEGFP and he Na+- ansloca ing mPPase
om M. mazei (MVP).
This s udy
and [28]
pIPP1GFP-699 2
Plasmid de i ed om pIPP1-699 bea ing he coding sequence o
gene IPP1 om S. ce e isiae ollowed in- ame by ha o yEGFP.
[24]
1
Plasmid used o complemen a ion and luo ide esis ance s udies.
2
Plasmid used o ans o ma ion me hod
based on selec ion in YPD.
Mic oo ganisms 2025,13, 226 6 o 18
2.8. Spon aneous Gene a ion o Fluo ide-Resis an Cells
W303-1A cells we e g own up o he exponen ial phase (OD
660
be ween 0.6 and 0.9)
in liquid unbu e ed YPD medium de oid o NaF; hen, en- old se ial dilu ions o hese
cul u es we e ca ied ou in s e ile wa e and 100
µ
L o each dilu ion was ex ended on o
2% (w/ ) aga pla es made in YPD bu e ed a pH 5 and supplemen ed wi h 60 o 70 mM
NaF. Pla es we e incuba ed a 30 ◦C o a leas one week.
2.9. T ans o ma ion o YPC3 Cells and Selec ion o T ans o man s in YPD
T ans o ma ion was pe o med by he me hod o Schies l and Gie z [
35
] wi h some
modi ica ions: 2 mL o YPGal was inocula ed wi h YPC3 cells om an aga pla e p epa ed
in he same medium and cul u ed o e nigh a 30
◦
C wi h agi a ion, as in Sec ion 2.3. The
ollowing day, 15 mL o YPGal was inocula ed wi h he o e nigh s a iona y cul u e so
ha he ini ial OD
660
was a ound 0.3. Cells we e g own o 4 h unde he same condi ions,
collec ed by cen i uga ion, esuspended in 15 mL o YPD, and g own o 2 mo e hou s
un il he OD660 was be ween 1 and 1.3. The las s ep was necessa y o allow YPC3 cells o
adap om galac ose o glucose me abolism be o e he ans o ma ion/selec ion p ocedu e.
Glucose-adap ed YPC3 cells we e collec ed by cen i uga ion (3000
×
g, 1 min in a
bench cen i uge), washed wi h 15 mL o s e ile wa e , esuspended in 1 mL o s e ile wa e ,
ans e ed o a mic o ube, and cen i uged again in a mic o uge (13,000
×
g, 30 s). The
inal washed pelle was esuspended in 1 mL o s e ile wa e , and 100 mL aliquo s we e
ans o med wi h plasmids pRS699b (con ol), pIPP1-699, pIPP1GFP-699 and piGMVP-
416 by he li hium ace a e/polye hylene glycol me hod [
35
]. A e hea -shock las ing
20–25 min a 42
◦
C, ans o med cells we e sedimen ed in a mic o uge and esuspended
in 1 mL o s e ile wa e , and 20
µ
L aliquo s we e ex ended on o YPD aga pla es
bu e ed wi h 50 mM MES-TRIS, pH 5, wi h he op ional addi ion o NaF (up o 40 mM
inal concen a ion).
2.10. Fluo escence Mic oscopy
Indi idual colonies o YPC3 ans o med as desc ibed in he p e ious pa ag aph
we e g own in liquid YPD o 4 h and di ec ly isualized wi h a ully au oma ed Leica
DM6000B mic oscope (Leica Mic osys ems) wi h FITC g een luo escence il e s (exci-
a ion il e 480/40 nm, dich oma ic mi o 505 nm, supp ession il e 527/30 nm), a
40
×
objec i e, and equipped wi h a cooled CCD (cha ge-coupled de ice) came a (ORCA-
AG, Hamama su Pho onics).
3. Resul s
YPC3 cells we e ans o med wi h plasmids bea ing gene coding o di e en e olu-
iona ily un ela ed PPases unde he con ol o cons i u i e p omo e s (Table 1).
T ans o man s eco e ed he capaci y o g ow unde e men a i e condi ions (2%
(w/ ) glucose) wi h he only excep ion being cells ans o med wi h plasmid pRS699b.
Mo eo e , YPC3 cells ans o med wi h plasmids pIPP1-699, pSPP2-699, pSPP2-416 and
piGMVP-416 could also g ow in he p esence o up o 75 mM NaF, unlike he pa en al s ain
W303-1A. In e es ingly, cells ans o med wi h he plasmids bea ing he gene coding o he
luo ide-insensi i e amily II sPPase om S. mu ans showed he highes ole ance o his
anion. Plasmids pIPP1-416 and pPPA1-426 we e less e ec i e a con e ing esis ance o
his sal (Figu e 1). Iden ical esul s we e ob ained wi h KF, demons a ing ha he oxici y
o NaF was a speci ic e ec o luo ide.
Mic oo ganisms 2025,13, 226 7 o 18
Mic oo ganisms 2025, 13, x FOR PEER REVIEW 7 o 19
Mo eo e , YPC3 cells ans o med wi h plasmids pIPP1-699, pSPP2-699, pSPP2-416 and
piGMVP-416 could also g ow in he p esence o up o 75 mM NaF, unlike he pa en al
s ain W303-1A. In e es ingly, cells ans o med wi h he plasmids bea ing he gene cod-
ing o he luo ide-insensi i e amily II sPPase om S. mu ans showed he highes ole -
ance o his anion. Plasmids pIPP1-416 and pPPA1-426 we e less effec i e a con e ing
esis ance o his sal (Figu e 1). Iden ical esul s we e ob ained wi h KF, demons a ing
ha he oxici y o NaF was a speci ic effec o luo ide.
Figu e 1. D op es s o ans o med YPC3 cells and pa en al s ain W303-1A g own a diffe en pH
alues and concen a ions o NaF unde e men a i e condi ions. YPC3 cells we e ans o med wi h
plasmids shown in Table 1 and g own as desc ibed in he main ex . Se ial dilu ions o he cul u es
we e p epa ed in s e ile wa e , spo ed on o YPD aga pla es con aining 50 mM MES (adjus ed o
he speci ied pH alues wi h TRIS) and supplemen ed wi h he indica ed concen a ions o NaF.
G ow h was eco ded a e 3 days, excep o pla es con aining 75 mM NaF, which we e g own o
4 days be o e he pho og aph was aken. Cell iabili y declined wi h NaF concen a ions abo e 75
mM un il he comple e hal ing o g ow h was obse ed a 100 mM NaF.
The esul s ob ained wi h YPD aga pla es we e con i med by g owing cells in liquid
cul u e media (Figu e 2). Fo he sake o cla i y, only he g ow h o W303-1A cells and
s ain YPC3 ans o med wi h plasmid pIPP1-699 in liquid YPD, pH 5.0, wi h 75 mM NaF
is shown, bu simila esul s we e ob ained wi h YPC3 cells ans o med wi h plasmids
pSPP2-699 and pSPP2-416.
Figu e 1. D op es s o ans o med YPC3 cells and pa en al s ain W303-1A g own a di e en pH
alues and concen a ions o NaF unde e men a i e condi ions. YPC3 cells we e ans o med wi h
plasmids shown in Table 1and g own as desc ibed in he main ex . Se ial dilu ions o he cul u es
we e p epa ed in s e ile wa e , spo ed on o YPD aga pla es con aining 50 mM MES (adjus ed o he
speci ied pH alues wi h TRIS) and supplemen ed wi h he indica ed concen a ions o NaF. G ow h
was eco ded a e 3 days, excep o pla es con aining 75 mM NaF, which we e g own o 4 days
be o e he pho og aph was aken. Cell iabili y declined wi h NaF concen a ions abo e 75 mM un il
he comple e hal ing o g ow h was obse ed a 100 mM NaF.
The esul s ob ained wi h YPD aga pla es we e con i med by g owing cells in liquid
cul u e media (Figu e 2). Fo he sake o cla i y, only he g ow h o W303-1A cells and
s ain YPC3 ans o med wi h plasmid pIPP1-699 in liquid YPD, pH 5.0, wi h 75 mM NaF
is shown, bu simila esul s we e ob ained wi h YPC3 cells ans o med wi h plasmids
pSPP2-699 and pSPP2-416.
Mic oo ganisms 2025, 13, x FOR PEER REVIEW 8 o 19
Figu e 2. G ow h o W303-1A cells and YPC3 cells ans o med wi h plasmid pIPP1-699 in liquid
YPD adjus ed o pH 5.0 in he absence and p esence o 75 mM NaF. (-■-) W303-1A cells g own
wi hou NaF, (-●-) YPC3 cells ans o med wi h plasmid pIPP1-699 wi hou NaF, (--♦--) W303-1A
g own wi h 75 mM NaF, and (- -▲- -) YPC3 cells ans o med wi h plasmid pIPP1-699 g own wi h
75 mM NaF. The OD
660
alues a e he a e ages o h ee independen expe imen s.
YPC3 cells ans o med wi h plasmids bea ing diffe en PPases also inc eased luo-
ide esis ance in aga pla es wi h medium con aining glyce ol, conside ed a ‘non- e -
men able’ ca bon sou ce in yeas [41] (Figu e 3).
Figu e 3. D op es s o ans o med yeas mu an YPC3 cells and pa en al s ain W303-1A g own a
diffe en pH alues and concen a ions o NaF unde espi a o y condi ions. Expe imen s we e pe -
o med as in Figu e 1, excep ha 3% (w/ ) glyce ol subs i u ed o glucose in he pla es.
C ude ex ac s om YPC3 cells ans o med wi h plasmid pIPP1-699 showed a 10-
old inc ease in soluble PPase-speci ic ac i i y as compa ed o hose ob ained om he
pa en al s ain W303-1A, he inc ease being only a ound 50% highe in he case o cells
ans o med wi h cen ome ic plasmid pIPP1-416. YPC3 cells ans o med wi h high-copy
Figu e 2. G ow h o W303-1A cells and YPC3 cells ans o med wi h plasmid pIPP1-699 in liquid YPD
adjus ed o pH 5.0 in he absence and p esence o 75 mM NaF. (-
■
-) W303-1A cells g own wi hou
NaF, (-
•
-) YPC3 cells ans o med wi h plasmid pIPP1-699 wi hou NaF, (--
♦
--) W303-1A g own wi h
75 mM NaF, and (- -
▲
- -) YPC3 cells ans o med wi h plasmid pIPP1-699 g own wi h 75 mM NaF.
The OD660 alues a e he a e ages o h ee independen expe imen s.
Mic oo ganisms 2025,13, 226 8 o 18
YPC3 cells ans o med wi h plasmids bea ing di e en PPases also inc eased luo ide
esis ance in aga pla es wi h medium con aining glyce ol, conside ed a ‘non- e men able’
ca bon sou ce in yeas [41] (Figu e 3).
Mic oo ganisms 2025, 13, x FOR PEER REVIEW 8 o 19
Figu e 2. G ow h o W303-1A cells and YPC3 cells ans o med wi h plasmid pIPP1-699 in liquid
YPD adjus ed o pH 5.0 in he absence and p esence o 75 mM NaF. (-■-) W303-1A cells g own
wi hou NaF, (-●-) YPC3 cells ans o med wi h plasmid pIPP1-699 wi hou NaF, (--♦--) W303-1A
g own wi h 75 mM NaF, and (- -▲- -) YPC3 cells ans o med wi h plasmid pIPP1-699 g own wi h
75 mM NaF. The OD
660
alues a e he a e ages o h ee independen expe imen s.
YPC3 cells ans o med wi h plasmids bea ing diffe en PPases also inc eased luo-
ide esis ance in aga pla es wi h medium con aining glyce ol, conside ed a ‘non- e -
men able’ ca bon sou ce in yeas [41] (Figu e 3).
Figu e 3. D op es s o ans o med yeas mu an YPC3 cells and pa en al s ain W303-1A g own a
diffe en pH alues and concen a ions o NaF unde espi a o y condi ions. Expe imen s we e pe -
o med as in Figu e 1, excep ha 3% (w/ ) glyce ol subs i u ed o glucose in he pla es.
C ude ex ac s om YPC3 cells ans o med wi h plasmid pIPP1-699 showed a 10-
old inc ease in soluble PPase-speci ic ac i i y as compa ed o hose ob ained om he
pa en al s ain W303-1A, he inc ease being only a ound 50% highe in he case o cells
ans o med wi h cen ome ic plasmid pIPP1-416. YPC3 cells ans o med wi h high-copy
Figu e 3. D op es s o ans o med yeas mu an YPC3 cells and pa en al s ain W303-1A g own
a di e en pH alues and concen a ions o NaF unde espi a o y condi ions. Expe imen s we e
pe o med as in Figu e 1, excep ha 3% (w/ ) glyce ol subs i u ed o glucose in he pla es.
C ude ex ac s om YPC3 cells ans o med wi h plasmid pIPP1-699 showed a
10- old inc ease in soluble PPase-speci ic ac i i y as compa ed o hose ob ained om
he pa en al s ain W303-1A, he inc ease being only a ound 50% highe in he case o cells
ans o med wi h cen ome ic plasmid pIPP1-416. YPC3 cells ans o med wi h high-copy
plasmid pPPA1-426, bea ing he gene coding o he human cy osolic sPPase, also showed
a signi ican inc ease in soluble PPase ac i i y (a ound h ee- old) wi h espec o W303-1A
cells. In all hese cases, he espec i e ac i i ies we e s ongly inhibi ed by he p esence o
0.5 mM NaF in he assay (Figu e 4).
Lowe le els o speci ic PPase ac i i y, be ween 5- and 10- old less han hose obse ed
wi h mul i-copy plasmids bea ing amily I sPPases, we e de ec ed in ex ac s ob ained om
cells ans o med wi h pSPP2-699 and pSPP2-416, which encode a amily II sPPase. In hese
cases, he soluble PPase ac i i y was insensi i e o luo ide (Figu e 4).
Cells ans o med wi h piGMVP-416, encoding he Na
+
- ansloca ing mPPase om M.
mazei (MVP), only showed memb ane-associa ed luo ide-insensi i e PPase ac i i y ha
inc eased by six- o se en- old in he p esence o 100 mM KCl, as p e iously desc ibed [
28
].
Immunode ec ion pe o med in cell ex ac s wi h a polyclonal an ibody agains S.
ce e isiae IPP1 showed ha , in YPC3 cells ans o med wi h plasmids pIPP1-699, pIPP1-416
and pPPA1-426, inc eases in PPase ac i i y co ela ed wi h he le els o a polypep ide o
32 kDa, he expec ed size o euka yo ic amily I sPPase polypep ides [
18
]. No band was
Mic oo ganisms 2025,13, 226 9 o 18
de ec ed in cells ans o med wi h con ol plasmid pRS699b o wi h pSPP2-699, pSPP2-416,
and piGMVP-416 (Figu e 5).
Mic oo ganisms 2025, 13, x FOR PEER REVIEW 9 o 19
plasmid pPPA1-426, bea ing he gene coding o he human cy osolic sPPase, also showed
a signi ican inc ease in soluble PPase ac i i y (a ound h ee- old) wi h espec o W303-
1A cells. In all hese cases, he espec i e ac i i ies we e s ongly inhibi ed by he p esence
o 0.5 mM NaF in he assay (Figu e 4).
Lowe le els o speci ic PPase ac i i y, be ween 5- and 10- old less han hose ob-
se ed wi h mul i-copy plasmids bea ing amily I sPPases, we e de ec ed in ex ac s ob-
ained om cells ans o med wi h pSPP2-699 and pSPP2-416, which encode a amily II
sPPase. In hese cases, he soluble PPase ac i i y was insensi i e o luo ide (Figu e 4).
Figu e 4. Le els o hyd oly ic sPPase ac i i y in p o ein ex ac s obse ed om ans o med yeas
mu an YPC3 cells and pa en al s ain W303-1A. Ac i i y assays we e pe o med in he absence
(black columns) and p esence (whi e columns) o 0.5 mM NaF, as desc ibed in he Sec ion 2. Values
o speci ic ac i i ies a e a e ages ± SE co esponding o a leas 5 independen expe imen s. S u-
den ’s unpai ed es s we e pe o med using he T es calcula o on he webpage
h ps://www.g aphpad.com/quickcalcs/ es 1/? o ma =SEM (accessed on 10 Janua y 2025). Legend:
(1) s a is ically signi ican diffe ence (p = 0.0133), (2) e y s a is ically signi ican diffe ence (p =
0.0042), (3) e y s a is ically signi ican diffe ence (p = 0.0024), (4) ex emely s a is ically signi ican
diffe ence (p = 0.0008), (5) e y s a is ically signi ican diffe ence (p = 0.0045), (6) e y s a is ically
signi ican diffe ence (p = 0.0070), and (7) s a is ically signi ican diffe ence (p = 0.0211).
Cells ans o med wi h piGMVP-416, encoding he Na
+
- ansloca ing mPPase om
M. mazei (MVP), only showed memb ane-associa ed luo ide-insensi i e PPase ac i i y
ha inc eased by six- o se en- old in he p esence o 100 mM KCl, as p e iously desc ibed
[28].
Immunode ec ion pe o med in cell ex ac s wi h a polyclonal an ibody agains S.
ce e isiae IPP1 showed ha , in YPC3 cells ans o med wi h plasmids pIPP1-699, pIPP1-
416 and pPPA1-426, inc eases in PPase ac i i y co ela ed wi h he le els o a polypep ide
o 32 kDa, he expec ed size o euka yo ic amily I sPPase polypep ides [18]. No band
was de ec ed in cells ans o med wi h con ol plasmid pRS699b o wi h pSPP2-699,
pSPP2-416, and piGMVP-416 (Figu e 5).
Figu e 4. Le els o hyd oly ic sPPase ac i i y in p o ein ex ac s obse ed om ans o med yeas
mu an YPC3 cells and pa en al s ain W303-1A. Ac i i y assays we e pe o med in he absence (black
columns) and p esence (whi e columns) o 0.5 mM NaF, as desc ibed in he Sec ion 2. Values o speci ic
ac i i ies a e a e ages
±
SE co esponding o a leas 5 independen expe imen s. S uden ’s unpai ed
es s we e pe o med using he T es calcula o on he webpage h ps://www.g aphpad.com/
quickcalcs/ es 1/? o ma =SEM (accessed on 10 Janua y 2025). Legend: (1) s a is ically signi ican
di e ence (p= 0.0133), (2) e y s a is ically signi ican di e ence (p= 0.0042), (3) e y s a is ically
signi ican di e ence (p= 0.0024), (4) ex emely s a is ically signi ican di e ence (p= 0.0008), (5) e y
s a is ically signi ican di e ence (p= 0.0045), (6) e y s a is ically signi ican di e ence (p= 0.0070),
and (7) s a is ically signi ican di e ence (p= 0.0211).
Mic oo ganisms 2025, 13, x FOR PEER REVIEW 10 o 19
Figu e 5. Immunode ec ion o amily I sPPases in p o ein ex ac s ob ained ans o med YPC3 cells
and pa en al s ain W303-1A (uppe panel). A polyclonal an ibody agains S. ce e isiae IPP1 was
used. In o al, 50 µg o o al p o ein was loaded pe lane. The lowe panel shows he Ponceau S
s aining o he ni ocellulose il e a e ans e ing he p o eins om he SDS-PAGE gel
.
In e nal le els o PPi signi ican ly inc eased in soluble ex ac s o W303-1A cells and
YPC3 cells ans o med wi h plasmid pIPP1-416 a e g owing o only 2 h in he p esence
o NaF. The o e exp ession o amily I PPases o exp ession o he S. mu ans amily II
PPase SPPA2 p e en ed his scena io (Figu e 6).
Figu e 6. In e nal le els o PPi measu ed in ans o med YPC3 cells and pa en al s ain W303-1A
a e 2 h o g owing in he p esence o 75 mM NaF (black columns). Whi e columns show PPi le els
o he espec i e con ol cells g own in s anda d YPD (see he Sec ion 2 o u he de ails o he
p ocedu e). Values a e a e ages ± SE co esponding o 4 independen expe imen s. S uden ’s un-
pai ed es s we e pe o med using he T es calcula o a on webpage
h ps://www.g aphpad.com/quickcalcs/ es 1/? o ma =SEM (accessed on 10 Janua y 2025) Legend:
(*) s a is ically signi ican diffe ence (p = 0.0457) and (**) e y s a is ically signi ican diffe ence (p =
0.0025).
Fluo ide- esis an yeas cells spon aneously appea ed a e he g ow h o W303-1A
cells o 6–7 days in YPD aga pla es buffe ed a pH 5 and supplemen ed wi h 70 mM NaF.
These expe imen s we e ca ied ou as desc ibed in he Sec ion 2 wi h six independen
W303-1A clones. A ound 0.003% o cells we e es ima ed o spon aneously de elop
Figu e 5. Immunode ec ion o amily I sPPases in p o ein ex ac s ob ained ans o med YPC3 cells
and pa en al s ain W303-1A (uppe panel). A polyclonal an ibody agains S. ce e isiae IPP1 was used.
In o al, 50
µ
g o o al p o ein was loaded pe lane. The lowe panel shows he Ponceau S s aining o
he ni ocellulose il e a e ans e ing he p o eins om he SDS-PAGE gel.
Mic oo ganisms 2025,13, 226 16 o 18
Abb e ia ions
The ollowing abb e ia ions a e used in his manusc ip :
IPP1 nucleocy osolic ino ganic py ophospha ase om S. ce e isiae
PPi ino ganic py ophospha e
PPase ino ganic py ophospha ase
m-PPase memb ane-bound ion- ansloca ing ino ganic py ophospha ase
MVP Na+- ansloca ing m-PPase om he a chaea Me hanosa cina mazei
OD660 op ical densi y a 660 nm
PPA1 cy osolic ino ganic py ophospha ase om Homo sapiens
sPPase soluble ino ganic py ophospha ases
SPP2 ino ganic py ophospha ase om S. mu ans
yEGFP yeas -enhanced g een luo escen p o ein
Re e ences
1.
Ba bie , O.; A eola-Mendoza, L.; Del Razo, L.M. Molecula Mechanisms o Fluo ide Toxici y. Chem. Biol. In e ac . 2010,188,
319–333. [C ossRe ] [PubMed]
2.
Smi h, F.A.; Hodge, H.C.; Dinman, B.D. Ai bo ne Fluo ides and Man: Pa I. C i . Re . En i on. Sci. Technol. 1977,8, 293–371.
[C ossRe ]
3.
Few ell, L.; Smi h, S.; Kay, D.; Ba am, J. An A emp o Es ima e he Global Bu den o Disease Due o Fluo ide in D inking
Wa e . J. Wa e Heal h 2006,4, 533–542. [C ossRe ] [PubMed]
4. B eake , R.R. New Insigh on he Response o Bac e ia o Fluo ide. Ca ies Res. 2012,46, 78–81. [C ossRe ]
5.
Bake , J.L.; Suda san, N.; Weinbe g, Z.; Ro h, A.; S ockb idge, R.B.; B eake , R.R. Widesp ead Gene ic Swi ches and Toxici y
Resis ance P o eins o Fluo ide. Science 2012,335, 233–235. [C ossRe ]
6.
S ockb idge, R.B.; Wacke , L.P. The Link be ween Ancien Mic obial Fluo ide Resis ance Mechanisms and Bioenginee ing
O gano luo ine Deg ada ion o Syn hesis. Na . Commun. 2024,15, 4593. [C ossRe ]
7.
McIlwain, B.C.; Rup ech , M.T.; S ockb idge, R.B. Memb ane Expo e s o Fluo ide Ion. Annu. Re . Biochem. 2021,90, 559–579.
[C ossRe ]
8.
Johns on, N.R.; Cline, G.; S obel, S.A. Cells Adap o Resis Fluo ide h ough Me abolic Deac i a ion and In acellula Acidi ica-
ion. Chem. Res. Toxicol. 2022,35, 2085–2096. [C ossRe ]
9.
Qin, J.; Chai, G.; B ewe , J.M.; Lo elace, L.L.; Lebioda, L. Fluo ide Inhibi ion o Enolase: C ys al S uc u e and The modynamics.
Biochemis y 2006,45, 793–800. [C ossRe ]
10.
Li, L. The Biochemis y and Physiology o Me allic Fluo ide: Ac ion, Mechanism, and Implica ions. C i . Re . O al Biol. Med. 2003,
14, 100–114. [C ossRe ]
11.
Heikinheimo, P.; Leh onen, J.; Bayko , A.; Lah i, R.; Coope man, B.S.; Goldman, A. The S uc u al Basis o Py ophospha ase
Ca alysis. S uc . Lond. Engl. 1993 1996,4, 1491–1508. [C ossRe ] [PubMed]
12. Lah i, R. Mic obial Ino ganic Py ophospha ases. Mic obiol. Re . 1983,47, 169–178. [C ossRe ] [PubMed]
13.
Bayko , A.A.; Malinen, A.M.; Luo o, H.H.; Lah i, R. Py ophospha e-Fueled Na+ and H+ T anspo in P oka yo es. Mic obiol. Mol.
Biol. Re . MMBR 2013,77, 267–276. [C ossRe ] [PubMed]
14.
Se ano, A.; Pé ez-Cas iñei a, J.R.; Bal sche sky, M.; Bal sche sky, H. H+-PPases: Yes e day, Today and Tomo ow. IUBMB Li e
2007,59, 76–83. [C ossRe ]
15.
Coope man, B.S.; Bayko , A.A.; Lah i, R. E olu iona y Conse a ion o he Ac i e Si e o Soluble Ino ganic Py ophospha ase.
T ends Biochem. Sci. 1992,17, 262–266. [C ossRe ]
16.
Shin ani, T.; Uchiumi, T.; Yonezawa, T.; Salminen, A.; Bayko , A.A.; Lah i, R.; Hachimo i, A. Cloning and Exp ession o a
Unique Ino ganic Py ophospha ase om Bacillus Sub ilis: E idence o a New Family o Enzymes. FEBS Le . 1998,439, 263–266.
[C ossRe ]
17.
Young, T.W.; Kuhn, N.J.; Wadeson, A.; Wa d, S.; Bu ges, D.; Cooke, G.D. Bacillus Sub ilis ORF yybQ Encodes a Manganese-
Dependen Ino ganic Py ophospha ase wi h Dis inc i e P ope ies: The Fi s o a New Class o Soluble Py ophospha ase?
Mic obiology 1998,144, 2563–2571. [C ossRe ]
18.
Gómez-Ga cía, M.R.; Losada, M.; Se ano, A. Compa a i e Biochemical and Func ional S udies o Family I Soluble Ino ganic
Py ophospha ases om Pho osyn he ic Bac e ia. FEBS J. 2007,274, 3948–3959. [C ossRe ]
19.
Kim, E.J.; Zhen, R.G.; Rea, P.A. He e ologous Exp ession o Plan Vacuola Py ophospha ase in Yeas Demons a es Su iciency o
he Subs a e-Binding Subuni o P o on T anspo . P oc. Na l. Acad. Sci. USA 1994,91, 6128–6132. [C ossRe ]
20.
Bayko , A.A.; Fab ichniy, I.P.; Pohjanjoki, P.; Zy yano , A.B.; Lah i, R. Fluo ide E ec s along he Reac ion Pa hway o Py ophos-
pha ase: E idence o a Second Enzyme.Py ophospha e In e media e. Biochemis y 2000,39, 11939–11947. [C ossRe ]
Mic oo ganisms 2025,13, 226 17 o 18
21.
Bayko , A.A.; Anashkin, V.A.; Salminen, A.; Lah i, R. Ino ganic Py ophospha ases o Family II—Two Decades a e Thei
Disco e y. FEBS Le . 2017,591, 3225–3234. [C ossRe ] [PubMed]
22.
Kolakowski, L.F.; Schloesse , M.; Coope man, B.S. Cloning, Molecula Cha ac e iza ion and Ch omosome Localiza ion o he
Ino ganic Py ophospha ase (PPA) Gene om S. Ce e isiae. Nucleic Acids Res. 1988,16, 10441–10452. [C ossRe ] [PubMed]
23.
Lundin, M.; Bal sche sky, H.; Ronne, H. Yeas PPA2 Gene Encodes a Mi ochond ial Ino ganic Py ophospha ase Tha Is Essen ial
o Mi ochond ial Func ion. J. Biol. Chem. 1991,266, 12168–12172. [C ossRe ] [PubMed]
24.
Se ano-Bueno, G.; Mad oñal, J.M.; Manzano-López, J.; Muñiz, M.; Pé ez-Cas iñei a, J.R.; He nández, A.; Se ano, A. Nuclea
P o easomal Deg ada ion o Saccha omyces Ce e isiae Ino ganic Py ophospha ase Ipp1p, a Nucleocy oplasmic P o ein Whose
S abili y Depends on I s Subcellula Localiza ion. Biochim. Biophys. Ac a Mol. Cell Res. 2019,1866, 1019–1033. [C ossRe ]
25.
Se ano-Bueno, G.; He nández, A.; López-Lluch, G.; Pé ez-Cas iñei a, J.R.; Na as, P.; Se ano, A. Ino ganic Py ophospha ase
De ec s Lead o Cell Cycle A es and Au ophagic Cell Dea h h ough NAD+ Deple ion in Fe men ing Yeas . J. Biol. Chem. 2013,
288, 13082–13092. [C ossRe ]
26.
D ake, R.; Se ano, A.; Pé ez-Cas iñei a, J.R. N-Te minal Chimae as wi h Signal Sequences Enhance he Func ional Exp ession
and Al e he Subcellula Localiza ion o He e ologous Memb ane-Bound Ino ganic Py ophospha ases in Yeas . Biochem. J. 2010,
426, 147–157. [C ossRe ]
27.
Pé ez-Cas iñei a, J.R.; He nández, A.; D ake, R.; Se ano, A. A Plan P o on-Pumping Ino ganic Py ophospha ase Func ionally
Complemen s he Vacuola ATPase T anspo Ac i i y and Con e s Ba ilomycin Resis ance in Yeas . Biochem. J. 2011,437, 269–278.
[C ossRe ]
28.
Pé ez-Cas iñei a, J.R.; Se ano, A. The H+-T ansloca ing Ino ganic Py ophospha ase F om A abidopsis Thaliana Is Mo e Sensi i e
o Sodium Than I s Na+-T ansloca ing Coun e pa F om Me hanosa cina Mazei. F on . Plan Sci. 2020,11, 1240. [C ossRe ]
29. Hanahan, D. S udies on T ans o ma ion o Esche ichia Coli wi h Plasmids. J. Mol. Biol. 1983,166, 557–580. [C ossRe ]
30. Thomas, B.J.; Ro hs ein, R. Ele a ed Recombina ion Ra es in T ansc ip ionally Ac i e DNA. Cell 1989,56, 619–630. [C ossRe ]
31.
Se ano, R.; Villalba, J.-M. Chap e 35 Exp ession and Localiza ion o Plan Memb ane P o eins in Saccha omyces. In Me hods in
Cell Biology; Galb ai h, D.W., Bou que, D.P., Bohne , H.J., Eds.; Academic P ess: Camb idge, MA, USA, 1995; Volume 50, pp.
481–496. ISBN 0091-679X.
32.
Siko ski, R.S.; Hie e , P. A Sys em o Shu le Vec o s and Yeas Hos S ains Designed o E icien Manipula ion o DNA in
Saccha omyces Ce e isiae. Gene ics 1989,122, 19–27. [C ossRe ] [PubMed]
33.
Mad oñal de Sancha, J.M. Es udios Funcionales de las Pi o os a asas Ino gánicas de Saccha omyces Ce e isiae y O ganismos
Fo osin é icos. Ph.D. Thesis, Uni e sidad de Se illa, Se illa, Spain, 2017.
34.
Ch is ianson, T.W.; Siko ski, R.S.; Dan e, M.; She o, J.H.; Hie e , P. Mul i unc ional Yeas High-Copy-Numbe Shu le Vec o s.
Gene 1992,110, 119–122. [C ossRe ] [PubMed]
35.
Schies l, R.H.; Gie z, R.D. High E iciency T ans o ma ion o In ac Yeas Cells Using Single S anded Nucleic Acids as a Ca ie .
Cu . Gene . 1989,16, 339–346. [C ossRe ] [PubMed]
36. T eco, D.A.; Lundblad, V. P epa a ion o Yeas Media. Cu . P o oc. Mol. Biol. 2001,23, 13.1.1–13.1.7. [C ossRe ]
37.
Ra hbun, W.B.; Be lach, M.V. Es ima ion o Enzymically P oduced O hophospha e in he P esence o Cys eine and Adenosine
T iphospha e. Anal. Biochem. 1969,28, 436–445. [C ossRe ]
38.
Val e de, F.; Losada, M.; Se ano, A. Func ional Complemen a ion o an Esche ichia Coli Gap Mu an Suppo s an Amphibolic
Role o NAD(P)-Dependen Glyce aldehyde-3-Phospha e Dehyd ogenase o Synechocys is Sp. S ain PCC 6803. J. Bac e iol. 1997,
179, 4513–4522. [C ossRe ]
39.
Heinonen, J.K.; Honkasalo, S.H.; Kukko, E.I. A Me hod o he Concen a ion and o he Colo ime ic De e mina ion o
Nanomoles o Ino ganic Py ophospha e. Anal. Biochem. 1981,117, 293–300. [C ossRe ]
40.
López-Ma qués, R.L. Fisiología Molecula y Bioquímica de Pi o os a asas T anslocado as de P o ons. Doc o al Disse a ion,
Uni e sidad de Se illa, Se illa, Spain, 2004. A ailable online: h ps://dialne .uni ioja.es/se le / esis?codigo=23243 (accessed
on 10 Janua y 2025).
41.
Aßkamp, M.R.; Klein, M.; Ne oig , E. Saccha omyces Ce e isiae Exhibi ing a Modi ied Rou e o Up ake and Ca abolism o
Glyce ol Fo ms Signi ican Amoun s o E hanol om This Ca bon Sou ce Conside ed as ‘Non-Fe men able’. Bio echnol. Bio uels
2019,12, 257. [C ossRe ]
42.
Ga cía-Con e as, R.; De La Mo a, J.; Mo a-Mon es, H.M.; Ma ínez-Ál a ez, J.A.; Vicen e-Gómez, M.; Padilla-Vaca, F.; Va gas-
Maya, N.I.; F anco, B. The Ino ganic Py ophospha ases o Mic oo ganisms: A S uc u al and Func ional Re iew. Pee J 2024,
12, e17496. [C ossRe ]
43.
Johns on, N.R.; S obel, S.A. Ni a e and Phospha e T anspo e s Rescue Fluo ide Toxici y in Yeas . Chem. Res. Toxicol. 2019,32,
2305–2319. [C ossRe ]
44.
Ma quis, R.E.; Clock, S.A.; Mo a-Mei a, M. Fluo ide and O ganic Weak Acids as Modula o s o Mic obial Physiology. FEMS
Mic obiol. Re . 2003,26, 493–510. [C ossRe ] [PubMed]
Mic oo ganisms 2025,13, 226 18 o 18
45.
Ka im, A.S.; Cu an, K.A.; Alpe , H.S. Cha ac e iza ion o Plasmid Bu den and Copy Numbe in Saccha omyces Ce e isiae o
Op imiza ion o Me abolic Enginee ing Applica ions. FEMS Yeas Res. 2013,13, 107–116. [C ossRe ] [PubMed]
46.
Niu, H.; Zhu, J.; Qu, Q.; Zhou, X.; Huang, X.; Du, Z. C ys allog aphic and Modeling S udy o he Human Ino ganic Py ophos-
pha ase 1: A Po en ial An i-cance D ug Ta ge . P o eins S uc . Func . Bioin o ma. 2021,89, 853–865. [C ossRe ]
47.
Diako , T.T.; Ta sio, M.; Kane, P.M. Measu emen o Vacuola and Cy osolic pH In Vi o in Yeas Cell Suspensions. J. Vis. Exp.
2013, 50261. [C ossRe ]
48.
O iss, I.R.; A ne , T.R.; Russell, R.G.G. Py ophospha e: A Key Inhibi o o Mine alisa ion. Cu . Opin. Pha macol. 2016,28, 57–68.
[C ossRe ]
49.
Addison, W.N.; Aza i, F.; Sø ensen, E.S.; Kaa inen, M.T.; McKee, M.D. Py ophospha e Inhibi s Mine aliza ion o Os eoblas
Cul u es by Binding o Mine al, Up-Regula ing Os eopon in, and Inhibi ing Alkaline Phospha ase Ac i i y. J. Biol. Chem. 2007,
282, 15872–15883. [C ossRe ]
50. Fang, S.; Fang, X. Ad ances in Glucose Me abolism Resea ch in Colo ec al Cance . Biomed. Rep. 2016,5, 289–295. [C ossRe ]
51.
Wang, P.; Zhou, Y.; Mei, Q.; Zhao, J.; Huang, L.; Fu, Q. PPA1 Regula es Tumo Malignan Po en ial and Clinical Ou come o Colon
Adenoca cinoma h ough JNK Pa hways. Onco a ge 2017,8, 58611–58624. [C ossRe ]
52.
Mish a, D.R.; Chaudha y, S.; K ishna, B.M.; Mish a, S.K. Iden i ica ion o C i ical Elemen s o Regula ion o Ino ganic Py ophos-
pha ase (PPA1) in MCF7 B eas Cance Cells. PLoS ONE 2015,10, e0124864. [C ossRe ]
53.
Wimme , J.L.E.; Kleine manns, K.; Ma in, W.F. Py ophospha e and I e e sibili y in E olu ion, o Why PPi Is No an Ene gy
Cu ency and Why Na u e Chose T iphospha es. F on . Mic obiol. 2021,12, 759359. [C ossRe ]
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