The Multiple Roles of Polyphosphate in Ralstonia eutropha and Other Bacteria

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]. This is an Open Access a icle licensed unde he C ea i e Commons A ibu ion-NonComme cial-4.0 In e na ional License (CC BY-NC) (h p://www.ka ge .com/Se ices/OpenAccessLicense), applicable o he online e sion o he a icle only. Usage and dis ibu ion o com- me cial pu poses equi es w i en pe mission. 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 165 Mic ob Physiol 2021;31:163–177 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. Rosigkei /Kneißle/Ob uča/Jend ossek Mic ob Physiol 2021;31:163–177 166 DOI: 10.1159/000515741 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 167 Mic ob Physiol 2021;31:163–177 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 Rosigkei /Kneißle/Ob uča/Jend ossek Mic ob Physiol 2021;31:163–177 168 DOI: 10.1159/000515741 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.