Resea ch A icle
Mic ob Physiol 2021;31:163–177
The Mul iple Roles o Polyphospha e in
Rals onia eu opha and O he Bac e ia
Hanna Rosigkei
a Lea Kneißle
a S anisla Ob uča
b Die e Jend ossek
a
aIns i u e o Mic obiology, Uni e si y o S u ga , S u ga , Ge many; bFacul y o Chemis y, B no Uni e si y o
Technology, B no, Czech Republic
Recei ed: Decembe 22, 2020
Accep ed: Ma ch 6, 2021
Published online: May 20, 2021
Co espondence o:
Die e Jend ossek, die e .jend ossek @ imb.uni-s u ga .de
© 2021 The Au ho (s)
Published by S. Ka ge AG, Basel
ka ge @ka ge .com
www.ka ge .com/mip
DOI: 10.1159/000515741
Keywo ds
Polyphospha e · Polyphospha e kinase · Rals onia eu opha ·
Cup ia idus neca o
Abs ac
An as onishing a ie y o unc ions has been a ibu ed o
polyphospha e (polyP) in p oka yo es. Besides being a ese -
oi o phospho us, unc ions in exopolysaccha ide o ma-
ion, mo ili y, i ulence and in su i ing a ious o ms o
s esses such as exposu e o hea , ex eme pH, oxida i e
agen s, high osmola i y, hea y me als and o he s ha e been
asc ibed o polyP. In his con ibu ion, we will p o ide a his-
o ical o e iew on polyP, will hen desc ibe he key p o eins
o polyP syn hesis, he polyP kinases, be o e we will c i ically
assess o he unde lying da a on he mul iple unc ions o
polyP and p o ide e idence ha – wi h he excep ion o a
P-s o age- unc ion – mos o he unc ions o polyP a e no
ele an o su i al o Rals onia eu opha, a bio echnologi-
cally impo an be a-p o eobac e ial species.
© 2021 The Au ho (s)
Published by S. Ka ge AG, Basel
In oduc ion
His o ical Backg ound
The p esence o insoluble phospha e-con aining in-
clusions in mic oo ganisms has been known o a long
ime. The i s desc ip ions go back o Babes [Babes 1885]
and Liebe mann [Liebe mann 1888] and we e i s sum-
ma ized by A. Meye in 1904 [Meye 1904]. PolyP is also
known as me aphospha e o olu in g anules (iden i ica-
ion o polyP in Spi illum olu ans) because o he me a-
ch omic shi o basic dyes upon binding o polyphos-
pha e [Wiame 1947]. PolyP was egula ly ound in yeas
[Wid a 1959] and in se e al bac e ial species such as
Ae obac e ae ogenes [Smi h e al. 1954] and in pa icula
in Mycobac e ium and Co ynebac e ium species [Sall e
al., 1958; Knaysi 1959]. I became e iden ha olu in
(polyP) g anules a e widesp ead in mic oo ganisms ( o
ea ly e iews see [Wid a 1959; Ha old 1966; Kulae and
Vagabo 1983], and meanwhile i is well accep ed ha
polyP is p esen in all kingdoms o li e [Ko nbe g e al.,
1999; Kulae and Kulako skaya 2000; Rao e al., 2009]
and p esumably is p esen in e e y species. Fo he mos
ecen e iews on polyP see [Albi and Se ano 2016; Jimé-
nez e al., 2016; Xie and Jakob 2018].
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Rosigkei /Kneißle/Ob uča/Jend ossek
Mic ob Physiol 2021;31:163–177
164
DOI: 10.1159/000515741
Func ions o PolyP
Many unc ions ha e been asc ibed o polyP. The mos
e iden one is ha o a s o age compound o phospho us
and he coun e -ions o he phospha e anions such as
Ca2+, Mg2+, K+. Howe e , addi ional unc ions ha e been
add essed o polyP: polyP can be a ese oi o ene gy
due o i s ene gy- ich phospho -anhyd ide bonds and
polyP can subs i u e ATP in polyP-dependen phospho -
yla ion (kinase) eac ions. PolyP is di ec ly o indi ec ly
in ol ed in pa hogenici y [Pa ks and Hobden 2005; Peng
e al., 2016; Kuma e al., 2016; S isanga e al., 2019] and
in mo ili y [Rashid e al., 2000; Shi e al., 2004; Zhang e
al., 2005; F aley e al., 2007; Hossain e al., 2008]. PolyP
can modula e he cellula esponses o a ious s esses
and is impo an o he adap a ion o s a iona y phase
condi ions [Rao and Ko nbe g 1996; Nikel e al., 2013;
Casey e al., 2013; G ay and Jakob 2015]. In Esche ichia
coli, polyP is pa o he s ingen esponse o nu ien
downshi s [Kulae and Vagabo 1983; Rao e al., 1998;
Ko nbe g e al., 1999; Ku oda e al., 1999; Kulae and Ku-
lako skaya 2000; Ku oda e al., 2001], ecen ly upda ed by
M. G ay [G ay 2019]. PolyP may be also in ol ed in he
con e sion o (pa hogenic) bac e ia in o he iable bu
non cul u able (VBNC) s a e [Gangaiah e al., 2009].
Resea ch o he las wo decades showed ha polyP is
in ol ed in and impo an o ole ance agains s esses
(hea , UV ligh , sol en s, neu ophilic oxidan s/hypoha-
lous acids, hea y me als, an ibio ics, su i al in s a iona y
phase and o he s) [Kim e al., 2002; F aley e al., 2007;
Seu e held e al., 2008; Va ela e al., 2010; Nikel e al.,
2013; Toche a e al., 2013; Alcán a a e al., 2014; G ay and
Jakob 2015; B u e al., 2017; G oi l e al., 2017; Sul ana e
al., 2020]. Compounds ha inhibi he o ma ion o polyP
in bac e ia (mesalamine = 5-amino-salicylic acid) educe
he polyP con en in in es inal bac e ia in mammalians
and in u n sensi ize hem o hos de ense by eac i e ox-
ygen species. This could explain he posi i e e ec o me-
salamine on pa ien s su e ing om ulce a i e coli is
[Dahl e al., 2017]. Recen ly published esul s p o ide e -
idence bo h o euka yo es (yeas ) and p oka yo es (Pseu-
domonas ae uginosa) ha polyP is necessa y o cell cycle
con ol, cell cycle exi and epai o DNA damage [B u e
al., 2016; Racki e al., 2017; B u e al., 2017]. PolyP has also
a p ominen ole in bac e ia ha a e pa o he biological
phospha e emo al p ocess (EBPR) in sewage sludge
[Blackall e al., 2002; Se iou e al., 2003; Yang e al.,
2017].
In p o eins, polyP can be co alen ly bound o lysine
esidues in a p ocess named polyphospho yla ion. This
pos ansla ional modi ica ion can modula e he p o ein
ac i i y simila ly as o he co alen modi ica ions [Aze-
edo e al., 2015; Aze edo and Saia di 2016]. Recen ly, i
was shown ha polyP can ha e a chape one-like unc ion
by binding o pa ially dena u a ed/un olded p o eins
and keeping hem in a e olding compe en s a e [G ay e
al., 2014]. In mammalians, polyP is pa o he blood co-
agula ion sys em (p e-ac i a ion o Hageman ac o XII
by binding o polyP). Fu he mo e, polyP can in e ac
wi h p o eins p esen in he b ain o mammalians. PolyP
is able o bind o p e o ms o α-synuclein ib ils he eby
p e en ing hei up ake by neu ones. α-Synuclein is a key
p o ein in neu odegene a i e diseases such as Alzheime
o Pa kinson [Yoo e al., 2018; Lempa and Jakob 2019;
Lempa e al., 2019] and can o m amyloid-like agg e-
ga es ha a e ypical indica ions occu ing p io o he
ecognizable ou b eak o such diseases. PolyP can p o ec
he o ganism om he o ma ion o pa hogenic amyloid
plaques by binding o pa ially un olded α-synuclein p o-
eins and p e en ing hem om u he mis olding. These
indings sugges ha polyP has a p ominen unc ion in
neu odegene a i e diseases.
PolyP Kinases A e he Key Enzymes o PolyP
Me abolism in P oka yo es
PolyP kinases (PPKs) ca alyze he e e sible o ma ion
o polyP by ans e o a γ-phospha e g oup om ATP (o
ano he NTP) o a g owing chain o polyP. The p esence
o an oligophospha e p ime is no necessa y o ini ia e
he eac ion. The i s isola ed and biochemically cha ac-
e ized PPK was ha o E. coli [Ahn and Ko nbe g 1990;
Kumble e al., 1996]. The PPK o E. coli is he p o o ype
o so-called ype 1 PPKs (PPK1s) which a e p o eins wi h
molecula masses o ≈80 kDa and a e composed o ou
domains (N- e minal domain (N), head domain (H), and
wo C- e minal domains (C1 and C2)) [Zhu e al., 2005].
A second ype o PPK (so-called PPK2s) has been iden i-
ied i s in P. ae uginosa [Zhang e al., 2002; Ishige e al.,
2002] and la e in many o he bac e ia. Cu en ly known
PPK2s mos ly ha e abou hal o he molecula masses o
PPK1s (35–40 kDa) and a e cha ac e ized by a so-called
PPK2-domain. PPK2 o P. ae uginosa p e e s he e e se
eac ion, i.e., he nucleo ide dikinase eac ion (NTP syn-
hesis om polyP and NDPs) and wo ks bes wi h GDP
compa ed o ADP and o he NDPs. The e o e, PPK2 o
P. ae uginosa migh ha e a unc ion o p o ide enough
GTP o example o exopolysacha ide biosyn hesis
[Ishige e al., 2002] and his migh be one eason o pol-
yP being impo an o i ulence. Phylogene ic analysis
o he amino acid sequences and compa ison wi h bio-
chemical p ope ies o isola ed PPK2s e ealed ha
Polyphospha e in Rals onia eu opha
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DOI: 10.1159/000515741
PPK2s can be di ided in o h ee subg oups [Mo omu a
e al., 2014]: membe s o subg oup I and II ca alyze he
phospho yla ion o nucleoside diphospha es and nucleo-
side monophospha es, espec i ely, while subg oup III
PPK2s can ca alyze bo h eac ions.
Analysis o p oka yo ic genome sequences e ealed
ha bac e ia can ha e ei he a ppk1 gene, a ppk2 gene o
bo h a ppk1 and ppk2. Many bac e ial species ha e e en
mul iple copies o ppk1 and ppk2 genes. Rals onia eu o-
pha, o example, has wo ppk1 genes (ppk1a, ppk1b) and
i e ppk2 genes (ppk2a – ppk2e) [Tumli sch e al., 2015].
The p esence o mul iple genes o bo h ypes o PPKs
sugges s ha hey ul ill di e en unc ions some o which
p esumably ha e no ye been iden i ied. Biochemically
cha ac e ized PPK2s ha e a low subs a e speci ici y and
accep bo h pu ine and py imidine nucleo ides [Mo o-
mu a e al., 2014]. PPK2c o R. eu opha is he mos un-
speci ic PPK among biochemically cha ac e ized PPKs
and accep s all na u al ibo- and desoxy ibonucleo ides
[Hildenb and e al., 2020]. The pu i ied p o ein is e en
able o o m mic oscopically de ec able polyP g anules in
i o om NTPs [Hildenb and e al., 2019]. Some PPKs,
in pa icula PPK2s, no only o m NTPs om polyP and
NDPs bu also ca alyze he o ma ion o oligo-phospho -
yla ed nucleosides wi h mo e han h ee phospha e uni s.
The i s example was PPK1 o E. coli ha ca alyzed he
o ma ion o guanosine e aphospha e om GDP and
polyP [Ku oda and Ko nbe g, 1997]. In 2019, Mo dho s
e al. showed ha he PPK2s o Meiococcus ube , Sino hi-
zobium melilo i, F ancisella ula ensis and Acine obac e
johnsonii o med e a- and pen a-phospho yla ed ade-
nosine om ADP and polyP [Mo dho s e al., 2019].
Ve y ecen ly, ano he PPK2, PPK2 o Ag obac e ium u-
me aciens, was ound o ca alyze he o ma ion o highly
phospho yla ed nucleosides up o he nona-phospha es
om any NDP in he p esence o polyP [F ank e al.,
2020]. The physiological unc ions, ha hese oligophos-
pho yla ed nucleosides migh ha e, a e no known.
PolyP Me abolism in R. eu opha
R. eu opha s ain H16 (al e na i e designa ion Cup i-
a idus neca o ) is a amous β-p o eobac e ium due o i s
abili y o g ow chemoli hoau o ophically (H2/CO2) and
o accumula e la ge amoun s o he “bioplas ic” poly(3-
hyd oxybu y a e) (PHB) [Pohlmann e al., 2006]. Ano h-
e in e es ing p ope y o R. eu opha is he p esence o
se en ppk genes in i s genome. A leas h ee PPKs, PPK1a,
PPK2b and PPK2c, con ibu e o he biosyn hesis o pol-
yP g anules and ou o hem (PPK1a, PPK2c, PPK2d and
PPK2e) a e associa ed wi h polyP g anules in i o as e-
ealed by usion analysis wi h he enhanced yellow luo-
escen p o ein (eYFP). PPK1b and PPK2b a e localized
nea one o he cell poles and o m luo escen oci (apa
om polyP g anules) when used o eYFP, whe eas eYFP-
PPK2a is soluble in he cy oplasm [Tumli sch e al., 2015].
Only one o he se en PPKs o R. eu opha, PPK2c, has
been biochemically cha ac e ized so a [Hildenb and e
al., 2019; Hildenb and e al., 2020] and showed a e y
b oad nucleo ide speci ici y. The main unc ion o PPK2c
p esumably is o eplenish GTP and o he NTP pools
du ing imes o enhanced demand on he expense o p e-
iously accumula ed polyP. The p ope ies o he o he
PPKs in R. eu opha a e cu en ly de e mined in ou lab-
o a o y. Se e al p o eins a e associa ed wi h polyP g an-
ules in i o in addi ion o he ou PPKs (Fig.1). These
a e Pp A, Pp B, PPI18 and PPI27: Pp A and Pp B bo h
ha e a so-called conse ed his idine alpha-helical domain
(CHAD) [Iye and A a ind 2002; Tumli sch and Jen-
d ossek 2017]. Exp ession o usions o pp A o pp B wi h
Fig. 1. Model o a polyP g anule in R. eu opha. P o eins a ached
o polyP a e symbolized as sphe es wi h p oposed designa ions.
The associa ion o ou polyP kinases (PPK1a, PPK2c, PPK2d,
PPK2e), o wo CHAD-mo i con aining p o eins (phosins Pp A,
Pp B) and o wo addi ional p o eins (PPI18, PPI27) wi h polyP
g anules has been demons a ed in i o. Pu a i e coun e -ions
(Mg2+, Ca2+, Mn2+, Mn2+, K+) o nega i ely cha ged polyP mole-
cules a e also indica ed. The p esence o Mn2+ ions in i o is spec-
ula i e bu – in i o – i has been demons a ed, ha he p esence
o Mn2+ ions e ec ua es he o ma ion o oligo-phospho yla ed
nucleo ides by PPK2c [Hildenb and e al., 2020]. The PPK kinases
PPK1b, PPK2a and PPK2b a e no associa ed wi h polyP g anules
in i o [Tumli sch e al., 2015]. PPK1a, PPK2b and PPK2c con-
ibu e o polyP syn hesis in R. eu opha. The sizes o he p o eins
a e enla ged ela i e o he polyP g anule o be e isibili y.
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Mic ob Physiol 2021;31:163–177
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ey p and s aining o he cells wi h 4′,6-diamidin-2-phe-
nylindole (DAPI) esul ed in cell-pole localized polyP
g anules wi h a ached eYFP-Pp A o eYFP-Pp B in con-
as o polyP g anules esiding in he middle egion o
he cell as usual. The same esul was ob ained when u-
sions o ey p wi h CHAD-encoding genes o o he p o eo-
bac e ia we e exp essed in R. eu opha. Since eYFP-
CHAD p o eins we e soluble in a polyP-de icien back-
g ound (R. eu opha in which all se en ppk genes had
been dele ed), hese esul s indica e ha CHAD p o eins
speci ically bind o polyP su aces and can di ec he pol-
yP g anules o he cell poles i hey a e o e exp essed (Pp
s ands o polyP a ge ing). The designa ion o polyP-as-
socia ed CHAD-mo i con aining p o eins as “phosins”
in analogy o phasins, ha a e polyhyd oxyalkanoa e
(PHA) associa ed p o eins wi hou ob ious ca aly ic
unc ion, has been sugges ed o Pp A and Pp B [Tum-
li sch and Jend ossek 2017]. Meanwhile, phosins we e
iden i ied independen ly in o he species and hei bind-
ing speci ici y o polyP has been demons a ed [Lo enzo-
O s e al., 2019; We en e al., 2019]. The unc ions o he
wo emaining polyP associa ed p o eins in R. eu opha
(PPI18 and PPI27; PPI s ands o polyP in e ac ing p o-
ein) is unknown. Bo h p o eins co-localize wi h polyP
g anules (as usion wi h eYFP) in some bu no in all
g ow h s ages [Tumli sch 2017].
The unusual high numbe o se en ppk genes in he
genome o R. eu opha sugges s ha polyP has mul iple,
p esumably mo e unc ions han only ha o a s o age
ma e ial o phospho us. We hough ha he excep ion-
ally high numbe o ppk genes p edes ina es R. eu opha
as an ideal species o s udy he di e en unc ions o pol-
yP by knocking ou ppk genes and o s udy he pheno-
ypic e ec s, and his was he basis o he p esen s udy.
E. coli was included o compa ison.
Resul s and Discussion
Fo ma ion o PolyP in R. eu opha and E. coli
PolyP g anules can be isualized by s aining cells wi h
DAPI and imaging a a DAPI-polyP-speci ic wa eleng h
o ≈515 nm (in compa ison o DAPI-DNA a ≈465 nm)
[Klau h e al., 2006]. R. eu opha o ms one o wo DAPI-
s ainable disc e e polyP g anules (Fig.2 o Tumli sch and
Jend ossek [2017]) when he cells a e g own unde op i-
mal supply wi h nu ien s (such as nu ien b o h medi-
um) and app op ia e physical pa ame e s (mode a e em-
pe a u e [30°C] and pH [≈7]). PolyP g anules in R. eu o-
pha cells a e loca ed in he cen al (nucleoid) egion. In
con as , E. coli is known o syn hesize polyP only unde
s ess condi ions such as nu ien downshi , ele a ed em-
pe a u e o oxida i e s ess [Rao e al., 1998; Ko nbe g e
al., 1999; G ay and Jakob 2015] bu he o ma ion o polyP
g anules in non-s essed E. coli wild- ype s ains has no
been demons a ed so a . To de e mine how polyP o -
ma ion in R. eu opha is in luenced by s ess condi ions,
we analyzed he o ma ion o polyP be o e and a e expo-
su e o di e en s ess condi ions. Fo mos expe imen s,
E. coli was used as a con ol. The expe imen s we e pe -
o med wi h bo h wild- ype (WT) s ains and he co e-
sponding polyP- ee mu an s ains (R. eu opha ∆ppk-all
in which all se en ppk genes had been dele ed and E. coli
∆ppk; E. coli has only one ppk gene [Akiyama e al., 1992]).
Hea Shock and G ow h a Ele a ed Tempe a u es
Ha e No E ec on PolyP Fo ma ion in R. eu opha
R. eu opha and E. coli (WT and ∆ppk mu an s) we e
g own on NB o LB medium a 30 and 37°C, espec i ely,
and he polyP con en s we e de e mined by luo escence
mic oscopy and polyP ex ac ion om lyophilized sam-
ples. PolyP de e mina ion was epea ed a e applying a
sho - ime (0–20 min) hea s ess (55°C). Figu e 2a shows
E. coli and R. eu opha cells s ained wi h DAPI be o e and
a e a hea s ess impulse. Disc e e DAPI-polyP oci we e
no de ec ed in E. coli cells unde any condi ion. Occa-
sionally, E. coli cells wi h di use DAPI signals in he cell
pe iphe y o nea he cell poles we e obse ed. When he
cells we e imaged in b igh ield, hea -s essed cells o en
e ealed da k bubble-like signals nea he poles and/o
he cell pe iphe y. Some o hese signals seemed o co-
localize wi h he DAPI signal. Howe e , he DAPI and he
b igh ield signals we e de ec ed no only in he WT bu
we e also ound in he ∆ppk mu an o he same ex en .
The e o e, hese signals a e unlikely o indica e he p es-
ence o polyP. They migh be a esponse o he cells o he
hea s ess and could ep esen agg ega es o hea -dam-
Fig. 2. a Fo ma ion o polyP in E. coli and in R. eu opha. E. coli
WT and E. coli Δppk (uppe le ) and R. eu opha WT and R. eu -
opha ∆ppk-all (uppe igh ) we e g own in LB o NB medium and
exposed o a 20 min o 10 min hea shock a 55°C, espec i ely, as
desc ibed in he Me hod sec ion. Samples aken be o e and a e
he hea shock we e s ained wi h DAPI and imaged in b igh ield
and in he DAPI-polyP channel. A ows poin o DAPI-s ained
polyP g anules. b PolyP o ma ion in E. coli WT and E. coli Δppk
(lowe le ) and R. eu opha WT and R. eu opha ∆ppk-all (lowe
igh ) upon incuba ion in MOPS-medium and exposu e o bleach.
Cells in inlay squa es we e added o he images o show a leas wo
indi idual cells pe condi ion. Mic oscopic pic u es show ep e-
sen a i e cells o wo independen expe imen s.
(Fo igu e see nex page.)
Polyphospha e in Rals onia eu opha
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DOI: 10.1159/000515741
WTΔppk-all
B igh ield DAPI-polyP Me geB igh ield
a
b
DAPI-polyP Me ge
WTΔppk
0
1 h con ol1 h HOCI
0
1 h con ol1 h HOCI
0
1 h con ol1 h HOCI 0
1 h con ol1 h HOCI
WTΔppk-all
B igh ield DAPI-polyP Me geB igh ield DAPI-polyP Me ge
WTΔppk
0
10 min10 min
0
0
20 min20 min 0
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2 µm 2 µm 2 µm
2

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aged cell componen s. In con as , R. eu opha WT cells
o med one o wo dis inc , globula -shaped g anule-like
s uc u es ha we e clea ly de ec able in he DAPI-polyP
channel. This was independen o whe he a hea s ess
impulse was applied o he cells o no . Globula , DAPI-
s ainable s uc u es we e no obse ed in he R. eu opha
∆ppk-all mu an unde any condi ion being in ag eemen
wi h he polyP-nega i e pheno ype o his mu an in ou
p e ious wo k [Tumli sch and Jend ossek 2017]. In sum-
ma y, ou da a indica e ha mic oscopically de ec able
polyP g anules a e no o med in E. coli ei he in hea -
s essed o in non-s essed cells bu ha polyP g anules
in R. eu opha WT a e o med independen o he appli-
ca ion o a hea -s ess impulse.
Nex , we di ec ly de e mined he amoun o o med
polyP in bo h species by polyP ex ac ion and subsequen
colo ime ic phospha e quan i ica ion. Non-s essed E.
coli cells (WT and he ∆ppk mu an ) did no con ain sig-
ni ican amoun s o polyP (<0.3 µmol Pi/g o cell d y
weigh [cdw]) (Table1). Ve y low amoun s o polyP o 1.0
± 0.4 µmol Pi/g cdw we e de ec ed in hea -s essed E. coli
WT cells, bu no signi ican polyP con en was de ec ed
in he ∆ppk mu an a e hea s ess. Appa en ly, E. coli is
able o syn hesize ace amoun s o polyP a e a hea -
s ess impulse, bu his amoun is oo low o be eliably
de ec ed by luo escence mic oscopy. In con as , much
highe le els o ≈60 µmol Pi/g cdw we e de e mined o
R. eu opha WT ha did only ma ginally inc ease o 65
(in NaCl) o 67 (in NB medium) µmol Pi/g cdw upon hea
s ess. In an independen epe i ion o his expe imen ,
we e en de e mined a sligh dec ease in he polyP con en
a e hea s ess (no shown). F om hese da a, we con-
clude ha he polyP le els in R. eu opha a e gene ally
much highe han in E. coli bu ha hea s ess does no
in luence he polyP le els o a la ge ex en in he wo spe-
cies. As expec ed, no polyP was de ec ed in he ∆ppk-all
mu an unde any condi ion and con i med he polyP-
nega i e pheno ype o he mu an s ain.
PolyP Does No Inc ease Su i al o R. eu opha a e
Hea S ess
To de e mine, whe he polyP could ha e an impac on
su i al o he bac e ia a e exposu e o a hea s ess im-
pulse, we de e mined su i al o WT cells and o he
polyP- ee ∆ppk-all mu an cells a e he hea s ess (0–
20 min 55°C). Howe e , we could no ind a signi ican
di e ence in he su i al o he WT e sus he polyP- ee
Table 1. PolyP le els in E. coli and in R. eu opha
S ain Medium polyP be o e hea shock
[µmol Pi/g cdw]
polyP a e hea shock
[µmol Pi/g cdw]
R. eu opha WT NB 60±3.3 67±2.4
R. eu opha WT 0.9% NaCl 60±3.3 65±0.7
R. eu opha ∆ppk-all 0.9% NaCl <0.3 <0.3
E. coli WT LB <0.3 1.0±0.4
E. coli WT 0.9% NaCl <0.3 <0.3
E. coli ∆ppk 0.9% NaCl <0.3 <0.3
R. eu opha: One biological eplica e wi h h ee echnical eplica es, E. coli in 0.9% NaCl a leas wo biologi-
cal eplica es, in LB, one biological eplica e, wi h h ee echnical eplica es.
1.00
0.10
0.01
0 2 4 6 8 10 12 14 16 18
Time, h
47 °
C
45 °
C
42 °
C
37 °
C
30 °C
OD600
WT
Δppk-all
Fig. 3. G ow h o R. eu opha WT and ∆ppk-all on NB medium. In
a mic o i e pla e, 200 µL o NB medium was inocula ed wi h an
18 h NB seed cul u e o R. eu opha WT (black ci cles) and ∆ppk-
all (open ci cles), espec i ely, in six echnical eplica es o an ini-
ial OD600 o abou 0.05. Cells we e cul i a ed a he indica ed em-
pe a u es o 15 h wi h cons an o bi al shaking. The g aphs dis-
play mean ± SD o six echnical eplica es.
Polyphospha e in Rals onia eu opha
169
Mic ob Physiol 2021;31:163–177
DOI: 10.1159/000515741
mu an (suppl. Fig. s1). When he su i al o E. coli cells
a e exposu e o a hea s ess impulse was es ed analo-
gously, E. coli WT and he ∆ppk mu an e ealed a simila
sensi i i y (suppl. Fig. s2). Only i highly dilu ed cell sus-
pensions o OD = 0.002 we e hea -challenged, E. coli WT
cells exhibi ed highe iabili y compa ed o he ∆ppk mu-
an . We do no know why he ∆ppk mu an was mo e
hea sensi i e a dilu ed cell concen a ions e sus high
cell concen a ions.
PolyP in R. eu opha Does No In luence G ow h a
High Tempe a u es
The polyP le els in R. eu opha did no change d a-
ma ically a e applica ion o a sho ime hea impulse as
shown abo e. To e alua e whe he polyP could be ad an-
ageous o g ow h a empe a u es nea he g empe a-
u e maximum we compa ed g ow h o R. eu opha WT
and he polyP- ee ∆ppk-all mu an a di e en empe a-
u es. As e iden om Figu e 3, no di e ence in g ow h
was de e mined be ween R. eu opha WT and he polyP-
ee ∆ppk-all mu an . Bo h s ains showed good g ow h
a 30–42°C bu could no mul iply a 45 o 47°C. A 42°C,
g ow h o bo h s ains was sligh ly educed compa ed o
37 o 30°C sugges ing ha 42°C is nea he uppe em-
pe a u e limi o R. eu opha. Howe e , he WT s ain
and he ∆ppk-all mu an showed he same g ow h cha -
ac e is ics a all es ed empe a u es. We conclude ha
polyP has no unc ion in hea ole ance in R. eu opha and
does no allow a be e g ow h a high empe a u es.
PolyP Does No Inc ease Oxida i e S ess Resis ance
Many epo s ha e p e iously desc ibed he bene icial
e ec s o polyP o esis ance o E. coli and o he species
agains oxida i e s ess such as hyd ogen pe oxide o
bleach [Casey e al., 2013; Dahl e al., 2017; G oi l e al.,
2017; Sul ana e al., 2020]. To es i oxida i e s ess has
an impac on su i al o R. eu opha, we exposed he cells
wi h 2 mM bleach (HOCl). E. coli cells we e also es ed o
compa ison. To his end, he WT and polyP- ee mu an
s ains o bo h species we e incuba ed in MOPS-bu e ed
medium supplemen ed wi h 1.3 mM phospha e and 0.2%
uc ose (R. eu opha) o 0.2% glucose (E. coli). A e in-
cuba ion o 3 h, he cells we e challenged wi h 2 mM
bleach o 15 min. Cell su i al was es ima ed by spo ing
app op ia e dilu ions on aga media and was addi ionally
moni o ed by he ac ion o cells ha we e esis an o
he up ake o p opidium iodide (PI). Al hough he num-
be o PI-posi i e R. eu opha cells inc eased upon expo-
su e o bleach, we could no de ec a signi ican di e ence
be ween WT and ∆ppk-all cells (Fig.4). This was in ag ee-
men wi h a compa able numbe o iable cells be ween
he WT and he ∆ppk-all mu an in he aga -spo assay
(no shown). Fo E. coli, howe e , we de e mined a sig-
ni ican di e ence be ween he WT and he ∆ppk-s ain
in sensi i i y owa d exposu e o bleach: HOCl- ea ed
cul u es o E. coli ∆ppk displayed a la ge inc ease in PI-
posi i e ∆ppk cells o ≈34%, whe eas he WT showed only
an inc ease om 5 o 9% PI-posi i e cells. The di e ence
in su i al be ween he WT and he ∆ppk mu an was
con i med by he aga -spo -assay (no shown).
Exposu e o Bleach Does No A ec he Fo ma ion o
PolyP G anules
To es whe he exposu e o bleach s imula es he o -
ma ion o polyP, we used he cells o he expe imen de-
sc ibed abo e o ollow he o ma ion o polyP g anules
Con ol 2 m
M
HOCl
0
10
20
30
40
50
% Pl posi i e cells
b
0
20
40
60
80
% Pl posi i e cells
Con ol 2 m
M
HOCl
a
■ WT
■ Δppk-all
■ WT
■ Δppk
Fig. 4. E ec o bleach (HOCl) on su i al
o R. eu opha and E. coli. R. eu opha WT
and ∆ppk-all (a) and E. coli WT and he
∆ppk mu an (b) we e cul i a ed in MOPS-
medium wi h 1.32 mM K2HPO4 and 0.2%
uc ose (o glucose in case o E. coli) a
30°C o 37°C and o bi al shaking. A e 6 h,
10 mL cell suspension was ans e ed o
esh lasks and ea ed wi h PBS (con ol)
o wi h 2 mM HOCl. Samples we e aken
a e 15 min, dilu ed in PBS and s ained
wi h p opidium iodide. Th ee biologic ep-
lica es wi h n = 10,000 e en s we e mea-
su ed by low cy ome y (a) o 2 biological
eplica es wi h 292 ≤ n ≤ 606 cells we e
coun ed by luo escence mic oscopy (b).
E o ba s show s anda d de ia ion.
Rosigkei /Kneißle/Ob uča/Jend ossek
Mic ob Physiol 2021;31:163–177
170
DOI: 10.1159/000515741
luo escence mic oscopically and o quan i y he amoun
o ex ac able polyP in cell samples. As shown in Figu e
2b, mos E. coli WT and R. eu opha WT cells o med one
( a ely wo) DAPI-s ainable polyP g anules du ing incu-
ba ion in he MOPS-phospha e-suga medium. In e es -
ingly, he numbe o DAPI-polyP g anules did no u he
inc ease upon exposu e o E. coli o R. eu opha WT cells
o bleach. As expec ed, he R. eu opha ∆ppk-all and ∆ppk
E. coli mu an s ains did no o m DAPI-s ainable polyP
g anules unde any condi ion. When he amoun o ex-
ac able polyP was de e mined, R. eu opha WT e ealed
a polyP con en o 45 µmol Pi/g cdw ha emained almos
cons an upon exposu e o bleach (Fig.5a). When E. coli
WT was es ed, a high polyP con en o 41 µmol Pi/g cdw
was de e mined in MOPS-phospha e-glucose medium
ha inc eased o 67 µmol Pi/g cdw a e exposu e o
bleach (Fig.5b). The o ma ion o mic oscopically de ec -
able polyP g anules and he de e mina ion o a high alue
o 41 µmol Pi/g cdw be o e ea men o E. coli WT wi h
bleach was unexpec ed as no polyP was de ec ed in cells
a e g ow h in LB medium (Fig.2a). We assume ha in-
cuba ion o E. coli cells in a phospha e- and ca bon sou ce-
con aining MOPS-medium ha lacks o he nu ien s en-
ables he cells o p oduce mo e ATP by espi a ion o glu-
cose han can be consumed by me abolism. The su plus
o ATP is ansien ly accumula ed in he o m o polyP
g anules. P esumably, he nu ien downshi om com-
plex medium o a MOPS-phospha e-glucose solu ion
p o oked s ess o he cells ha lead al eady o Dsk-de-
penden [G ay 2019] and ppGpp-associa ed polyP o -
ma ion (and/o inhibi ion o polyP deg ada ion by exo-
polyphospha ase) so ha he addi ion o bleach a a la e
ime-poin only ma ginally u he inc eased he o ma-
ion polyP. These indings sugges ha polyP has he
unc ion o a ba age o ATP du ing pe iods o a an-
sien misbalance o ATP-p oducing and ATP-consuming
eac ions.
PolyP Has No Impac on he Mo ili y o R. eu opha
PolyP has an impac on mo ili y o se e al bac e ial
species such as P. ae uginosa, Pseudomonas sy ingae, Ba-
cillus ce eus o Myxococcus xan hus [Rashid e al., 2000;
Shi e al., 2004; Zhang e al., 2005; F aley e al., 2007; Hos-
sain e al., 2008]. To analyze i his is also ue o R. eu -
opha, we de e mined mo ili y o R. eu opha WT and he
∆ppk-all mu an in swimming and swa ming assays.
Howe e , we could no ind any di e ence be ween he
WT and he ∆ppk-all mu an (suppl. Fig. s3). We con-
clude ha polyP has no impac on mo ili y in R. eu opha.
G ow h and Fo ma ion o PolyP in Mine al Sal s
Medium wi h o wi hou Phospha e.
Su p isingly, he p esence o polyP in R. eu opha WT
had no de ec able ad an age o su i al in ou expe i-
men s, and we could no de ec a pheno ype o he ∆ppk-
all mu an unde any o he es ed condi ions shown
abo e (hea shock/high empe a u e, eac i e oxygen spe-
cies, mo ili y). Appa en ly, polyP has no ob ious unc ion
in coping wi h hese s esso s. The indings sugges ha
polyP mus ha e ano he unc ion in R. eu opha. The
mos ob ious one is he unc ion as a ese oi o phos-
pho us. R. eu opha was o iginally isola ed om he
sp ing o he Wende i e nea Gö ingen/Ge many
[Wilde 1962]. The concen a ions o nu ien s such as ni-
ogen o phospho us sou ces a e usually low in aqua ic
ecosys ems and a e e en lowe in he sp ings o mos i -
1 mM
HOCI
Con ol 0
0
20
40
60
80
µmol Pi/g cdw
1 mM
HOCI
Con ol 0
0
20
40
60
µmol Pi/g cdw
■ WT
■ Δppk-all
■ WT
■ Δppk
ba
Fig. 5. E ec o bleach on he polyP le els
in R. eu opha and E. coli. R. eu opha WT
and ∆ppk-all (a) and E. coli WT and he
∆ppk mu an (b) we e incuba ed o 3 h in
a MOPS-medium wi h 1.32 mM K2HPO4
and 0.2% uc ose (o 0.2% glucose in case
o E. coli) a 30°C and 37°C espec i ely. A
= 0 50 mL cell suspensions o each s ain
we e ans e ed o esh lasks and ea ed
wi h PBS (con ol) o wi h 1 mM HOCl.
Samples we e aken a e 60 min.
Polyphospha e in Rals onia eu opha
171
Mic ob Physiol 2021;31:163–177
DOI: 10.1159/000515741
e s. The e o e, i makes sense o R. eu opha o s o e an
excess o phospho us in acellula ly in he o m o polyP
g anules, and his migh be one explana ion why polyP is
egula ly ound in s a iona y cells o R. eu opha cul u es
ha a e no limi ed by phospho us. To de e mine whe h-
e he p esence o polyP has an ad an age o R. eu opha
unde condi ions o limi ed nu ien supply, we es ed
g ow h, polyP con en , cell numbe s and cell iabili y in
a mine al sal s medium (MSM) wi h es ic ed supply o
phospho us. To his end, we cul i a ed R. eu opha WT
and he ∆ppk-all mu an in a modi ied MSM medium
wi h 0.8% uc ose in which phospha e, which is usually
p esen in high concen a ion as a bu e , was eplaced by
is(hyd oxyme hyl)aminome hane (T is). Phospha e
was added a 0.02% (1.3 mM) (Fig.6) o was comple ely
absen (Fig.7). A selec ed ime poin s, samples we e ak-
en and analyzed o op ical densi y (OD), polyP and PHB
con en . R. eu opha WT and he ∆ppk-all mu an g ew
compa ably well on T is-MSM in he p esence o uc ose
and 0.02% phospha e and eached OD600 alues o ≈5 a -
e 2 days (Fig.6a). Only in he exponen ial g ow h phase
be ween 12 and 36 h, he OD600 alues o he ∆ppk-all
6 12 18 24 36 48
Time, h
0
20
40
60
80
% PHB/g cdw
c
b
0 6 24 32 48
Time, h
0
20
40
60
80
100
µmol Pi/g cdw
■ WT
■ Δppk-all
a
120 24 36 48 60 72
Time, h
0.01
0.10
1.00
10.00
OD600
WT
Δppk-all
Fig. 6. G ow h o R. eu opha WT and ∆ppk-all on T is-bu e ed
mine al sal s medium wi h 0.8% uc ose and 0.02% KH2PO4 as
only ca bon and phospho us sou ces. Bo h cul u es we e inocu-
la ed wi h washed cells ob ained a e wo subsequen NB cul u es
(30°C, o e nigh and hen o 24 h). a Op ical densi y a 600 nm
(OD600). b PolyP con en by means o phospha e esidues (Pi) pe
g o cellula d y weigh (cdw) was de e mined by exopolyphospha-
ase-diges ion o isola ed polyP and colo ime ic measu emen
wi h an an imony- a a e-asco ba e assay. c PHB con en pe g
cdw was de e mined by gas ch oma og aphy a e acidic me hano-
lysis.