FtsZ phosphorylation pleiotropically affects Z-ladder formation, antibiotic production, and morphogenesis in Streptomyces coelicolor

Vol.: (0123456789) 1 3 An onie an Leeuwenhoek (2023) 116:1–19 h ps://doi.o g/10.1007/s10482-022-01778-w ORIGINAL PAPER F sZ phospho yla ion pleio opically a ec s Z‑ladde o ma ion, an ibio ic p oduc ion, andmo phogenesis inS ep omyces coelicolo PaulaYagüe · Joos Willemse · XianshaXiao · LeZhang · AngelMan eca · GillesP. anWezel Recei ed: 3 Janua y 2022 / Accep ed: 27 Augus 2022 / Published online: 16 No embe 2022 © The Au ho (s) 2022 de elopmen , colony mo phology, spo e esis ance, and an ibio ic p oduc ion in F sZ knockou mu an s exp essing F sZ alleles mimicking Se 319 and Se 387 phospho yla ion and non-phospho yla ion: AA (no phospho yla ion), AE, EA (mixed), and EE (double phospho yla ion). The F sZ-eGFP AE, EA and EE alleles we e no able o o m obse able F sZ-eGFP ladde s when hey we e exp essed in he S. coelicolo wild- ype s ain, whe eas he AA allele could o m appa en ly no mal eGFP Z-ladde s. The F sZ mu an exp essing he F sZ EE o EA o AE alleles is able o spo ula e indica ing ha he mu an alleles a e able o o m unc ional Z- ings leading o spo ula ion when he wild- ype F sZ gene is absen . The ou mu an s we e pleio opically a ec ed in colony mo phogen- esis, an ibio ic p oduc ion, subs a e mycelium di - e en ia ion and spo ula ion (spo ula ion iming and spo e esis ance) which may be an indi ec esul o he e ec in spo ula ion Z-ladde o ma ion. Each mu an showed a dis inc i e pheno ype in an ibio ic p oduc ion, single colony mo phology, and spo ula- ion (spo ula ion iming and spo e esis ance) indi- ca ing ha he di e en F sZ phosphomime ic alleles led o di e en pheno ypes. Taken oge he , ou da a p o ide e idence o a pleio opic e ec o F sZ phos- pho yla ion in colony mo phology, an ibio ic p oduc- ion, and spo ula ion. Keywo ds Cell di ision· Di e en ia ion· Se ine- phospho yla ion· Spo ula ion· S ep omyces Abs ac The GTPase F sZ o ms he cell di ision sca old in bac e ia, which media es he ec ui men o he o he componen s o he di isome. S ep o- myce es unde go wo di e en o ms o cell di i- sion. Sep a wi hou de ec able pep idoglycan di ide he highly compa men alised young hyphae du ing ea ly ege a i e g ow h, and c oss-walls a e o med ha dissec he hyphae in o long mul inucleoid com- pa men s in he subs a e mycelium, while ladde s o sep a a e o med in he ae ial hyphae ha lead o chains o uninucleoid spo es. In a p e ious s udy, we analysed he phosphop o eome o S ep omy- ces coelicolo and showed ha F sZ is phospho yl- a ed a Se 317 and Se 389. Subs i u ing Se –Se o ei he Glu–Glu (mimicking phospho yla ion) o Ala–Ala (mimicking non-phospho yla ion) hin ed a changes in an ibio ic p oduc ion. He e we analyse Supplemen a y In o ma ion The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi. o g/ 10. 1007/ s10482- 022- 01778-w. P.Yagüe(*)· J.Willemse· X.Xiao· L.Zhang· G.P. anWezel Depa men o Molecula Bio echnology, Ins i u e o Biology Leiden, Leiden Uni e si y, PO Box9505, 2300ABLeiden, TheNe he lands e-mail: yaguepaula@unio i.es A.Man eca Depa amen o de Biología Funcional e IUOPA, Á ea de Mic obiología, Facul ad de Medicina, Uni e sidad de O iedo, 33006O iedo, Spain 2 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) In oduc ion S ep omyce es a e ilamen ous bac e ia wi h a com- plex mul icellula li ecycle (Claessen e  al. 2014). A e ge mina ion, he hyphae g ow ou ia ip g ow h and b anching, o ming an in ica e ne wo k called he ege a i e mycelium. This ege a i e o subs a e mycelium unde goes se e al ounds o p o- g ammed cell dea h, which is equi ed o he o - ma ion o he ep oduc i e ae ial hyphae (Man eca e al. 2006; Yague e al. 2016). E en ually, he ae ial hyphae di e en ia e in o chains o spo es (Fla dh and Bu ne 2009). The biochemical pa hways egula ing his complex way o g ow h ha e no ye been ully cha ac e ized (Claessen e al. 2006; McCo mick and Fla dh 2012). S ep omyce es p oduce a wide ange o bioac i e me aboli es ha a e impo an o medicine, bio echnology, and ag icul u e (Ba ka e  al. 2016; Be dy 2005; Ka z and Bal z 2016). They a e e e ed o as na u e’s medicine make s (Hopwood 2007) and play a key ole in he p o ec ion o euka yo ic hos s agains challenges by pa hogens ( an Be geijk e al. 2020). The p oduc ion o seconda y me aboli es is closely linked o he de elopmen al p og am o s ep- omyce es ( an de Heul e al. 2018). Unde s anding he egula o y ne wo ks ha con ol he physiology o his in iguing bac e ium is one o he keys o unde - s anding and imp o ing seconda y me abolism ac i- a ion (Man eca and Yague 2018). Bac e ial cell di ision has been s udied ex en- si ely, whe eby much in o ma ion has been ob ained pa icula ly om he od-shaped bac e ia Esche ichia coli o Bacillus sub ilis (Du and Lu kenhaus 2017; E ing on and Wu 2017). These bac e ia g ow by elonga ion o he la e al wall and di ide by bina y ission (Koch 2000). The p ocess in ol es mo e han 35 p o eins ha o m he so-called di isome a mid- cell. F sZ is a ubulin-like GTPase p o ein (de Boe e al. 1992) ha polyme izes in o p o o ilamen s ha oge he o m he Z- ing (Sun and Ma golin 1998). The Z- ing is a cy oskele al s uc u e ha o ms he sca old a he si e o di ision and c ea es he con- s ic ing o ce ha e en ually di ides he cell in o wo daugh e cells (Vicen e e  al. 2006). The o ma ion o he Z- ing is egula ed nega i ely by he Min p o- eins ha p e en di ision a he cell poles (Szwed- ziak and Ghosal 2017; Howa d 2004), and nucleoid occlusion o p e en he o ma ion o he sep um o e non-seg ega ed ch omosomes (Ma golin 2004; Wu and E ing on 2004). Va ious F sZ-in e ac ing p o- eins ha e been disco e ed ha o en in e ac wi h he conse ed C- e minal end o he p o ein, showing ha his is a ho spo o p o ein in e ac ions (Huang e al. 2016). These in e ac ions play a majo ole in he polyme iza ion and co ec localiza ion o he Z- ing. The conse ed C- e minal pa (CCTP) o F sZ in e ac s among o he s wi h he memb ane e h- e s F sA and ZipA (Hale and de Boe 1997; Picho and Lu kenhaus 2002), he Z- ing s abilising p o eins ZapA (Low e al. 2004) and ZapD (Du and-He edia e al. 2012), and wi h SepF, which is he only one o hese p o eins conse ed in S ep omyces and o ms ing-like s uc u es ha p omo e F sZ polyme iza ion (Hamoen e al. 2006; K ol e al. 2012; Singh e al. 2008). Con e sely, he F sZ- ec ui ing SsgB in S ep- omyces in e ac s wi h he N- e minal pa o F sZ (Willemse e  al. 2011). The p ecise F sZ dynamics in bac e ial cell di ision and he iden i y o he di i- some p o eins a e s ill no ully unde s ood ( e iewed in (Ba ows and Goley 2021). Cell di ision in he mycelial s ep omyce es is coo dina ed di e en ly, in ol ing wo ypes o cell di ision (Jakimowicz and an Wezel 2012). Du ing ege a i e g ow h, cell di ision esul s in c oss-walls ha di ide he hyphae in o long mul inucleoid syncy - ial cells. Complex memb ane assemblies he eby o m ch omosome- ee zones in he hyphae du ing sep- um o ma ion, appa en ly p o ec ing he DNA om damage du ing di ision (Celle e al. 2016). Du ing spo ula ion-speci ic cell di ision in he ae ial hyphae, up o a hund ed sep a a e laid down mo e o less simul aneously, e en ually esul ing in he o ma ion o chains o uninucleoid spo es (Jakimowicz and an Wezel 2012). Vege a i e and spo ula ion-speci ic cell di ision also di e mechanis ically, as illus a ed by he ac ha many cell di ision genes ha a e equi ed o spo ula ion (e.g. sI, sL, and sW) a e dispensa- ble o ege a i e c oss-wall o ma ion (Benne e al. 2007, 2009; Mis y e al. 2008; Yague e al. 2016). Indeed, canonical cell di ision in ol ing he di isome is only seen du ing spo ula ion. While in mos bac e- ia cell di ision is nega i ely con olled, in s ep o- myce es F sZ is ac i ely ec ui ed by he SsgB p o- ein (Willemse e al. 2011). In u n, he localiza ion o SsgB depends on i s o hologue SsgA (T aag and an Wezel 2008), while SsgB is e he ed o he mem- b ane by SepG (Zhang e al. 2016). Thus he con ol o cell di ision di e s subs an ially om od-shaped 3 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) bac e ia, mos likely due o he absence o a mid- cell e e ence (Jakimowicz and an Wezel 2012). F sZ is essen ial in mos bac e ia, bu su p isingly, sZ mu an s o S ep omyces a e iable (McCo mick e al. 1994; Dai and Lu kenhaus 1991). Dynamin-like p o eins DynA and DynB we e epo ed o s abilise Z- ings du ing S ep omyces spo ula ion (Schlimpe e al. 2017). Recen wo ks disco e ed no el p o eins associa ed wi h S ep omyces F sZ du ing he ege- e a i e (Bush e al. 2022) and spo ula ion di isomes (Ramos-Leon e al. 2021). F sZ is also in ol ed in he o ma ion o sep a wi hou de ec able pep idoglycan in he highly compa men alised young S ep omyces hyphae o med a e spo e ge mina ion and p eced- ing he di e en ia ion o he mul inuclea ed subs a e mycelium (Yague e al. 2016), making S ep omyces cell di ision e en mo e complex. Besides he a ious laye s o ansc ip ional egu- la ion ha con ol he majo p ocesses in cells, pos - ansla ional modi ica ions (PTMs) also play a majo ole. P o ein phospho yla ion is one o he mos impo an PTMs in cells. His/Asp phospho yla ion is a well-known PTM in p oka yo es, since i o ms pa o he wo-componen sys ems, while Se /Th /Ty phospho yla ion is one o he mos impo an PTMs in euka yo es (Pe icko a and Pe icek 2003). How- e e , Se /Th /Ty phospho yla ion also exis s in bac- e ia, whe e i has impo an egula o y oles, hough i is s ill less well unde s ood han in euka yo es (Yague e al. 2019; Pe ei a e al. 2011; Pe icko a and Pe icek 2003). Membe s o he genus S ep omyces ha e one o he la ges phosphop o eomes desc ibed o da e (Pa ke e al. 2010; Man eca e al. 2011; Rios- e as e al. 2018). The S. coelicolo genome encodes some 34 Se ine Th eonine Kinase p o eins (STKs) and a leas 184 phosphop o eins (Hempel e al. 2012; Pa ke e  al. 2010; Riose as e al. 2018; Pe icko a and Pe icek 2003; Man eca e  al. 2011; Hi aka a e  al. 2019). To da e, he biological ele ance o S ep omyces Se /Th /Ty p o ein phospho yla- ion was only expe imen ally alida ed o Di IVA, an essen ial p o ein ha con ols pola g ow h and hyphal b anching (Hempel e  al. 2012) and DnaA, also an essen ial p o ein con olling DNA eplica ion (Lebkowski e al. 2020). Di IVA was also epo ed o be modula ed by phospho yla ion in S ep ococ- cus suis (Ni e al. 2018). Impo an cellula bac e ial p ocesses we e epo ed o be modula ed by Se ine/ Th eonine/Ty osine phospho yla ion as cell-wall emodelling, quo um sensing o bac e ial i ulence [ e iewed in Yague e al. (2019)]. The ac i i y o F sZ was epo ed o be modula ed by S/T/Y phospho yla- ion in some bac e ia as Deinococcus adiodu ans (Mau ya e  al. 2018) and Mycobac e ium ube cu- losis (Thaku and Chak abo i 2006). Despi e his knowledge, much mo e wo k will be necessa y o ully cha ac e ise and unde s and he biological ole o S ep omyces phosphop o eome and o he bac e ial phosphop o eomes. In a p e ious sho gun quan i a i e phosphop o- eomic s udy, we disco e ed 131 phosphop o eins in S. coelicolo , one o which was F sZ (Man eca e al. 2011; Riose as e al. 2018). To in es iga e he impo ance and biological ele ance o he F sZ phospho yla ions, we mu a ed esidues Se 319 and Se 387 simul aneously, c ea ing mu an s mimicking F sZ double phospho yla ion (F sZ-EE) and non- phospho yla ion (F sZ-AA). P elimina y analysis e ealed ha hese mu a ions had an e ec on second- a y me abolism (Riose as e al. 2018). In he cu en wo k, we u he analyse he biological e ec o F sZ phospho yla ion in he F sZ-EE and F sZ-AA mu an s and in wo new mu an s mimicking single F sZ phos- pho yla ion a Se 319 o Se 387 (Mu an s F sZ-EA and F sZ-AE) (Fig.1). We disco e ed ha , in addi- ion o seconda y me abolism, F sZ phospho yla ion shows a su p ising pleio opic pheno ype a ec ing Z-ladde o ma ion du ing spo ula ion, colony mo - phogenesis, spo ula ion iming, spo e mo phology, and spo e esis ance. To he bes o ou knowledge, his is he i s ime ha se ine phospho yla ion was desc ibed o in e e e wi h F sZ polyme isa ion and o a ec biological p ocesses di e en om seconda y me abolism. Ma e ial andme hods Bac e ial s ains and media S ep omyces coelicolo A3(2) M145 was ob ained om he John Innes Cen e (UK) s ain collec- ion (Kiese 2000). S ep omyces coelicolo M145 was he pa en o sZ* mu an s ains, F sZ-EE (pGlu319 and pGlu387; EE), F sZ-AA (pGlu319 and Ala387; EA), F sZ-EA (Ala387 and pGlu387; AE), and F sZ-AE (Ala319 and Ala387; AA). Esche ichia coli ET dam− 123,567 ha bou ing he 4 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) conjuga i e plasmid pUZ8002 (Page e  al. 1999) was used as hos o conjuga ion. SFM (soya la- ou , manni ol) aga medium (Hobbs 1989) was used as a spo ula ion medium and o he s udy o pheno ypes using scanning elec on mic oscopy, ansmission elec on mic oscopy o s e eo mic os- copy. GYM medium (glucose 5 g/l, yeas ex ac 4g/l, mal ex ac 5g/l, 0.5g/l MgSO4·7H2O, aga 20g/l and supplemen ed o a inal concen a ion o 0.5g/l K2HPO4) wi h cellophane disks was used as he g ow h medium o he con ocal obse a ions. MM (aga minimal medium) (Hopwood 1967), wi h manni ol as a ca bon sou ce, was used as a g ow h medium o checking he p oduc ion o pig- men s (an ibio ics) and he capaci y o spo ula ion. Fo S ep omyces, aga pla es we e inocula ed wi h 100ml o a suspension o 108 spo es pe ml, ol- lowed by incuba ion a 30°C. Fo E. coli, Lysogen b o h [LB, (Be ani 2004)] liquid/solid medium supplemen ed wi h 20% o glucose was used ol- lowed by incuba ion a 37°C. E. coli s ains ha bou ing he pBluesc ip II SK + and pNG3 plasmids we e g own in ampicillin (100μg/ml) amended media. Nalidixic acid (25μg/ ml) was used in he E. coli/S ep omyces conjuga ion o inhibi E. coli (Kiese 2000). S ep omyces s ains ha bou ing he in eg a i e pNG3 plasmid (Gonzalez- Quinonez e  al. 2016) we e g own in SFM supple- men ed wi h ap amycin (25 μg/ml) o spo ula ion. In o de o p e en an ibio ic in e e ences, he phe- no ypic analyses o he s ains exp essing he F sZ alleles cloned in o pNG3 we e pe o med wi hou an ibio ic. Cons uc ion o sZ* mu an s ains Di e en e sions o ecombinan sZ* whe eby he codons o Se 319 and Se 387 we e eplaced by codons o ei he glu ama e o alanine o combina- ions he eo we e syn he ized by GeneCus (www. genec us . com) and cloned in o pBluesc ip II SK + . Fo his, DNA agmen s we e ampli ied by PCR Fig. 1 Scheme o F sZ and he wo se ine si es objec o his s udy. A The amino acid sequence o F sZ p o ein. The ca oon ep esen s he F sZ co e s uc u e (in ed) om Mycobac e ium ube culosis (PDB ID 2Q1X). The linke egion o S ep omy- ces coelicolo F sZ spans esidues 315–389 (in blue), while he C- e minal ail (CTT) spans esidues 389–399 (in g een), which a e all depic ed by balls. The side chain o Se 319 and Se 387 a e labeled in ed s icks. B F sZ amino acid subs i u- ions in he mu an s. Subs i u ions o he 319 and 387 se ine si es a e highligh ed in ed. + (Glu) mimics pe manen phos- pho yla ion; − (Ala) mimics pe manen lack o phospho yla- ion 5 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) om he S. coelicolo M145 ch omosome using p ime s: F sZ_F and F sZ_R (Table 1). In eg a i e ec o pNG3 (Gonzalez-Quinonez e al. 2016) o i s de i a i es pNG3-EE, -EA, -AE, and -AA ha bou - ing a single copy o each ecombinan sZ* gene (exp essed om he na i e sZ p omo e egions desc ibed in (Fla dh e al. 2000) (Table1) we e in o- duced in o S. coelicolo M145 ia conjuga ion using E. coli ET12567/pUZ8002 as a dono s ain and ol- lowing he p o ocol desc ibed in Kiese e al. (2000). This gene a ed s ains ha bou ing con ol plasmid o exp essing F sZ-EE, -EA, -AE, o -AA. Subse- quen ly, he na i e sZ gene was inac i a ed using C isp -Cas9 me hodology in he ou di e en ans- conjugan s (F sZ-EE, F sZ-AA, F sZ-EA, F sZ-AE) elimina ing he sZ ORF and gene a ing he same sZ null backg ound in all he phospho yla ion/non- phospho yla ion mimicking mu an s (Tong e  al. 2015). In he same way, a con ol s ain ha bou ing sZ in pNG3 plasmid and subsequen ly inac i a ing he na i e copy, was pe o med demons a ing ha he pheno ypes o he mu an s a e no due o sZ placemen (Supplemen a y Fig.1A). Fo C isp -Cas9 he a ge sequence CGA TGA CTT TGA TGA CTG CG was used (p o ided by h p:// s a . biosu s ain. d u. dk/ laeb/ c ispy_ scoeli), he p ime sgRNA-F (NcoI) and he p ime sgRNA-R (SnaBI) (Table1). Fo he o e lapping ex emes ( ecombining agmen ) he ollowing p ime s we e used o allow homologous egion eplacemen , as desc ibed (Tong e al. 2015): 2082Le F, 2082Le R, 2082Righ F, 2082Righ R (Table 1). This esul ed in he dele ion o 1144 bp o sZ (n posi ions 2,234,455–2,235,599) om he S. coelicolo genome (Tong e al. 2015). As a con- ol, wild ype s ain in his s udy is ha bou ing emp y plasmid pNG3 (Table1). Cell iabili y and mo phology obse a ions a he con ocal mic oscope Fo he analysis o hypha iabili y, mo phology and spo ula ion iming, cul u es we e g own on GYM, ha es ed a di e en ime poin s om cellophane- g own mycelia, s ained wi h he LIVE/DEAD Bac- Ligh Bac e ial Viabili y Ki (In i ogen, L-13152) and obse ed unde he con ocal mic oscope ollow- ing ou p e ious p o ocol (Man eca e al. 2005). The LIVE/DEAD BacLigh Bac e ial Viabili y Ki con- sis s in d y SYTO 9 and P opidium Iodide (PI) which we p epa ed a he concen a ions ecommended by In i ogen, 6 µM and 30 µM espec i ely (bo h we e p epa ed in ul apu e mQ wa e ). Cellophane squa es (1cm side) we e manually cu , placed o e he mic oscope slide, 20 µL o he SYTO9/PI mix u e was added, a co e glass was ca e ully pu ed o e he sample p e en ing bubble o ma ion, he sample was incuba ed a oom empe a u e o 5min, and imme- dia ely obse ed a he mic oscope. This ki uses SYTO9 and P opidium Iodide (PI), wo DNA-binding colo an s. SYTO9 pene a es in ac memb anes and s ains iable cells g een, whe eas PI (s aining ed) only pene a es bac e ia wi h damaged memb anes. PI displaces SYTO9 om DNA when bo h colo an s a e p esen in dying cells. Samples we e obse ed using an in e ed Zeiss Axio Obse e Z lase scan- ning mic oscope a wa eleng hs o 488 and 568nm o exci a ion and 530 (g een) o 630 nm ( ed) o emission. Fo he analysis o sep a o ma ion, squa e mic os- copy co e glasses we e posi ioned in SFM pla es unde an angle o 45°, and subsequen ly, 10μl o a spo e suspension we e inocula ed (108 spo es/ml). A e 3–5days (depending on he s ain) o incuba- ion a 30°C, he co e glass was emo ed and 5µg/ ml o WGA-Alexa luo 633 we e added o cell-wall (pep idoglycan) s aining. Samples we e obse ed using an in e ed Leica SP8 lase scanning mic o- scope a wa eleng hs o 632 nm o exci a ion and 647nm o emission. Uns ained samples (p ocessed wi h ul apu e mQ wa e ins ead SYTO9/PI o WGA-Alexa luo ) we e used as con ols o ix he PMT gain le els a which au o luo escence was de ec ed, which we e much highe han he gain used o collec pic u es. Au o- luo escence, was ex emely low, compa ed o he s ained samples. A leas h ee biological eplica es we e p ocessed o each sample. Scanning elec on mic oscopy (SEM) Scanning elec on mic oscopy (SEM) was ca - ied ou as desc ibed (Colson e al. 2008). Fo his, small blocks o S ep omyces cul u es g own on SFM medium we e ixed using glu a aldehyde, dehyd a ed and d ied. E en ually, he samples in 100% ace one we e comple ely d ied in a c i ical poin d ye . Cells we e moun ed on an SEM s ub and spu e -coa ed wi h pla inum palladium o cap u e he images a 6 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) Table 1 Bac e ial s ains, plasmids and p ime s used in his s udy Desc ip ion Re e ences Bac e ial s ains Esche ichia coli TOP10 Ha bou ing he pBluesc ip II SK In i ogen® S ep omyces coelicolo M145 SCP1-SCP2-ha bou ing emp y plasmid pNG3 Kiese (2000) S ep omyces coelicolo M145 pGWS1574 ( habou ing sZunde i s own p omo e on pNG3) This s udy E. coli ET12567/pUZ8002 E. coli ET12567 con aining plasmid pUZ8002, a no sel - ansmissible plasmid which can mobilize o he plasmids; ClnR, KmR Fle e al. (1997), MacNeil e al. (1992) Plasmids pNG3 Cloning ec o ; HygR, AmpRGonzalez-Quinonez e al. (2016) pBluesc ip II SK Cloning ec o ; AmpRAgilen ® pCR™-Blun II-TOPO® Ze o Blun ®TOPO®PCR Cloning Ki , KmRIn i ogen® pHJL401 Cloning ec o ; Ts RLa son and He shbe ge (1986) pCRISPR-Cas9 Conjuga i e and he mosensi i e plasmid ha bou ing Cas9 Tong e al. (2015) pCRISPR-SgF sZ pCRISPR-Cas9 ha bou ing he a ge SCO2082 sequence and a 2kb agmen su ounding he sZ ORF used o knockdown sZ This s udy F sZ*-EE pNG3 pNG3 ha bo ing sZ* pGlu319 and pGlu387; Hyg R, AmpR, HygRRiose as e al. (2018) F sZ*-AA pNG3 pNG3 ha bo ing sZ* pGlu319 and Ala387; Hyg R, AmpR, HygRRiose as e al. (2018) F sZ*-EA pNG3 pNG3 ha bo ing sZ* Ala387 and pGlu387; HygR, AmpR, HygRThis s udy F sZ*-AE pNG3 pNG3 ha bo ing sZ* Ala319 and Ala387; HygR, AmpR, HygRThis s udy F sZ*-EE eGFP pHJL401 pHJL401 ha bou ing sZ*EE-eg p This s udy F sZ*-AA eGFP pHJL401 pHJL401 ha bou ing sZ*AA-eg p This s udy F sZ*-EA eGFP pHJL401 pHJL401 ha bou ing sZ*EA-eg p This s udy F sZ*-AE eGFP pHJL401 pHJL401 ha bou ing sZ*AE-eg p This s udy P ime s SCO4848F CGT CGT ATC CCC TCG GTT G Gonzalez-Quinonez e al. (2016) pMS82R GAG CCG GGA AAG CTC ATT CA Gonzalez-Quinonez e al. (2016) F sZ_F GGA CTA GTA GCA GGG TGT GCG GAA G This s udy F sZ_R AAG ATA TCC TAT CAC TTC AGG AAG TCCG This s udy sgRNA-F (NcoI) CAT GCC ATG GCG ATG ACT TTG ATG ACT GCG GTT TTA GAG CTA GAA ATA GC This s udy sgRNA-R (SnaBI) ACG CCT ACG TAA AAA AAG CAC CGA CTC GGT GCC This s udy 2082Le F AGG CCT AGA CCG ACC ACC GCC GAG This s udy 2082Le R CCT ATC ACT TCA GGA AGT CCG TGA TGA CTG CGA GGT AGT TCT G This s udy 2082Righ F CAG AAC TAC CTC GCA GTC ATC ACG GAC TTC CTG AAG TGA TAG G This s udy 2082Righ R AGG CCT AGT AAC CGA CCA CGG AAC GCA This s udy eGFP-FTSZ* F GTC AGA ATT CAG GCC TTC GAC GTG GCA GCA CCG CAG AAC TAC C This s udy eGFP-FTSZ* R GTC AAA GCT TGG ATC CTT CAG GAA GTC CGG CAC GTC C This S udy 7 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) 15kV. SEM pic u es we e used o measu ing spo e leng h. The leng hs o mo e han 250 spo es we e measu ed in each s ain using he ROI manage plugin o ImageJ. S a is ical signi icance was measu ed by compa ing spo e leng hs in wild- ype and he ou mu an s using a T- es . Th ee biological eplica es; 3 pla es/mu an , and h ee me hodological eplica es; 3 blocks pe pla e we e used o SEM expe imen s. T ansmission elec on mic oscopy (TEM) Fo isualizing he spo e chains o S ep omyces, small blocks we e cu om SFM pla es ha es ed in con luen cul u es and p ocessed o TEM essen ially as desc ibed (Pie e e al. 2005). The mycelium was washed wi h 1 × PBS be o e ixa ion wi h 1.5% glu- a aldehyde, and blocks we e hen pos - ixed wi h 1% osmium e oxide o 30min. The samples we e dehy- d a ed by passing hem h ough an e hanol g adien and placed in p opylene oxide o 15min ollowed by incuba ion in a mix u e o Epon and p opylene oxide (1:1) and pu e Epon (each s ep 45min). Finally, he samples we e embedded in Epon and sec ioned in o 70nm slices, which we e placed on 200-mesh cop- pe g ids. Samples we e s ained using u anyl-430 ace a e (2%) and lead ci a e (0.4%), i necessa y, and imaged a 70kV in a Jeol 1010 ansmission elec on mic oscope. Th ee biological eplica es; 3 pla es/mu an , and h ee me hodological eplica es; 3 blocks pe pla e we e used o SEM expe imen s. S e eo mic oscopy Single colonies o S. coelicolo and he mu an s ains we e obse ed using a Leica M80 s e eomic oscope. Pic u es we e aken wi h a Leica DFC295 came a. An ibio ic measu emen s Undecylp odigiosin and ac ino hodin we e quan i ied spec opho ome ically acco ding o Tsao e al. (1985) and Bys ykh e al. (1996). Cells we e up u ed in he cul u e medium by adding 0.1N KOH. A e o ex- ing and cen i uga ion, ac ino hodin was quan i ied in he supe na an (ɛ640 = 25,320). Undecylp odigiosin was measu ed a e acuum d ying o he mycelium, ollowed by ex ac ion wi h me hanol, acidi ica ion wi h HCl ( o 0.5M), and a spec opho ome ic assay (ɛ530 = 100,500). Rep oducibili y has been co obo- a ed by a leas h ee independen cul u es. Resis ance o spo es o lysozyme, hea ing, and eezing Fo hea ing (55°C, 30min) and eezing (− 20°C, 24h) shock ea men s, suspensions o 106 spo es/ml we e p epa ed in s e ile dis illed wa e and subjec ed o di e en ea men s as de ailed below. Ge mina ion o he spo es be o e and a e ea men was analysed by pla ing se e al dilu ions and quan i ying he num- be o colony- o ming uni s (Riose as e al. 2016). All quan i ica ions we e measu ed in iplica e. The da a co espond o he a e age ± SD o he eplica es. Fo lysozyme esis ance, 106 spo es/ml we e pla ed in LB aga medium and di ec ly placing il e discs con ain- ing 50μg, ollowing he me hod o Kleinschni z e al. (2011), and incuba ed a 30°C o 3days. sZ-eGFP alleles c ea ion and obse a ion a he con ocal and luo escence mic oscopes The pHJL401 plasmid (La son and He shbe ge 1986) (Table1), an E. coli/S ep omyces shu le ec- o , was used o he cons uc ions o he ou di e en sZ* (Se ine modi ica ions) and he eg p exp ession unde he con ol o he sZ p omo e . The sZ p o- mo e is cloned be ween si es EcoRI-S uI, he sZ* alleles wi hou s op codon a e cloned S uI-BamHI and he eg p is cloned downs eam BamHI-No I. The s ains wi h he eGFP usion we e analyzed by Axio Obse e Zeiss con ocal mic oscope. Exci a- ion was pe o med wi h a 488nm lase , and de ec- ion was pe o med wi h a 505–530 nm bandpass il e . The i s 16h o g ow h, we e obse ed mak- ing ime-lapse expe imen s as ollows; cul u es we e p e-g own on GYM medium o 6h a 30°C o he ge mina ion o he spo es, samples we e hen excised ou and in e ed in o µ-dishes (Ibidi GmbH 35mm, high ibi ea ). Pic u es we e aken e e y 10min du - ing 16 h ollowing Yagüe e  al. (2016). Wild- ype cul u es (wi hou F sZ-eGFP usions) we e used as con ols o ix he PMT gain le els a which au o- luo escence was de ec ed, which we e much highe han he gain used o collec he pic u es. Fo analys- ing he F sZ-eGFP a la e ime poin s, co e glasses we e posi ioned in SFM pla es unde an angle o 45°, and subsequen ly, 10μl o a spo e suspension we e 8 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) inocula ed (108 spo es/ml). A he indica ed ime- poin s, co e glasses we e emo ed, moun ed wi h ul apu e mQ wa e , and obse ed using a Leica DMRXA luo escence mic oscope wi h a FITC il e . Pic u es we e aken wi h an ORCA-Flash4.0 V3 Digi- al CMOS came a. Wild- ype s ain cul u es (wi hou F sZ-eGFP usions) we e used con ols o ix he le - els a which au o luo escence was de ec ed. A leas h ee biological eplica es we e p ocessed o each sample. Phase con as images mic oscopy Fo analysing he spo ula ion o F sZ*s ains, co e glasses we e posi ioned in SFM pla es unde an angle o 45°, and subsequen ly, 10 μl o a spo e suspen- sion we e inocula ed (108 spo es/ml). A e 5–7days, depending on he s ain, co e glasses we e emo ed, moun ed wi h ul apu e mQ wa e , and obse ed using phase con as unde he Leica DMRXA mic o- scope. Pic u es we e aken wi h an ORCA-Flash4.0 V3 Digi al CMOS came a. Image p ocessing Mic oscopy images we e p ocessed (his og am in en- si y le els we e adjus ed and scale added) using he Fiji so wa e (Schindelin e  al. 2012). Figu e com- posi es we e made using AdobePho osop CS5.1 and Adobe Pho oshop 2021. Resul s Cons uc ion o S ep omyces coelicolo s ains ha bou ing sZ* alleles mimicking F sZ phospho yla ion/non-phospho yla ion S ep omyces coelicolo F sZ (SCO2082) is di e en- ially phospho yla ed a Se 319 and Se 387 du ing he cell cycle (Riose as e al. 2018; Man eca e al. 2011). These se ine esidues a e loca ed in he p o ein linke , jus be ween he global domain and he C- e minal pa o F sZ (Fig.1A). Wi h he aim o analysing he e ec o F sZ se ine-phospho yla ion in S. coelicolo , we used a well-es ablished me hodology based on he subs i u ion o he Se esidues by Glu o Ala, hus mimicking phospho yla ion o non-phospho yla ion, espec i ely (Zhao e  al. 1994; Kelle -Pin e e  al. 2017; Hewi e al. 2017; Mo ison e al. 2003; T u - nye a e  al. 2005). Fou di e en mu an a ian s we e s udied: wo mu an s p e iously c ea ed, namely F sZ-EE (EE; mimicking wo phospho yla ions) and F sZ-AA (AA; mimicking non-phospho yla ion) (Riose as e  al. 2018); and wo new mu an s mim- icking phospho yla ion in only one o he se ines, namely F sZ-EA Glu-Ala (EA; mimicking phospho- yla ion a Se 319 and no phospho yla ion a Se 387), and F sZ-AE Ala-Glu (AE; mimicking no phospho- yla ion a Se 319 and phospho yla ion a Se 387) (Fig. 1B). The s ains, each exp essing one o he F sZ a ian s, we e gene a ed by he in oduc ion o he mu an copies in o an sZ null backg ound. Fo his, he ecombinan sZ*— ansc ibed om he na i e sZ p omo e egion (Fla dh e al. 2000)—was in oduced in o in eg a i e ec o pNG3 (Gonzalez- Quinonez e al. 2016) and in oduced in o S. coeli- colo M145. The na i e sZ was subsequen ly inac- i a ed using C isp -Cas9. As de ailed below, each mu an showed a dis inc i e pheno ype in an ibio ic p oduc ion, single colony mo phology and spo ula- ion (spo ula ion iming and spo e esis ance) indica - ing ha he di e en F sZ phosphomime ic alleles led o di e en pheno ypes. Phospho yla ion a ec s F sZ Z-ladde o ma ion du ing spo ula ion. To s udy Z- ing and Z-ladde o ma ion (1µm spaced Z- ings), we ollowed he me hodology de eloped by G an cha o a e al. (2005), whe e sZ-eGFP is ec op- ically exp essed in a S. coelicolo s ain ca ying he na i e sZ gene. This app oach was demons a ed o be use ul o o m unc ional F sZ-eGFP Z- ings ha can be obse ed du ing de elopmen and was la gely eplica ed in S ep omyces [see o ins ance Willemse and an Wezel (2009), Yague e al. (2016), Bush e al. (2022)].We exp essed F sZ-eGFP usions om he na i e sZ p omo e egion as p e iously desc ibed (G an cha o a e al. 2005). This was done o all ou mu an s (exp essing F sZ-AA, AE, EA and EE) and o wild- ype s Z. The cons uc s we e in oduced in o he S. coelicolo M145 and Z- ing o ma ion was analysed using luo escence mic oscopy (Fig.2). Du - ing S ep omyces de elopmen , he e a e wo s ages showing massi e Z- ing o ma ion. The i s one du - ing he ea ly de elopmen ( he i s 16h cul u e) co - esponding o he ea ly compa men alised mycelium 9 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) Fig. 2 Z- ings and ladde s o ma ion in he sZ*- eGFP alleles. Fluo escence mic og aphs o he young compa men alised hyphae (g owing on GYM), sub- s a e and ae ial mycelium (g owing on SFM) and spo ula ing hyphae (g ow- ing on SFM) a e shown. Con as mode images a e shown o he F sZ-EE, F sZ-EA, and F sZ-AE mu an s o illus a e ha hey a e spo ula ing. Only F sZ-AA o m spo ula ion Z-ladde s. Rep esen a i e images om a leas h ee biological eplica es a e shown. A ows indica e Z- ings. A oheads indica e F sZ-eGFP accumula ions ha do no c oss he ans- e se axis o he hyphae and do no cons i u e ma u e Z- ings. Scale ba s 10µm 16 An onie an Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) Au ho con ibu ions PY, JW, and XX pe o med he expe - imen s. PY made he igu es. PY, AM, and GPVW planned he expe imen s and w o e he manusc ip . Funding Open Access unding p o ided hanks o he CRUE-CSIC ag eemen wi h Sp inge Na u e. PY was unded by a Ma ie-Cu ie-Cla ín co und ellowship (FICYT, “Conseje ía de Educación y Ciencia, P incipado de As u ias, Spain”). Wo k in AM’s lab was unded by “Minis e io de Ciencia, Inno ación Uni e sidades/Agencia Es a al de In es- igación/Fondo Eu opeo de Desa ollo Regional” (P ojec RTI2018-093978-B-I00), and he “Conseje ía de Empleo, Indus ia y Tu ismo del P incipado de As u ias” (P ojec FC-GRUPIN-IDI/2018/000120). Decla a ions Con lic o in e es The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Open Access This a icle is licensed unde a C ea i e Com- mons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea- i e Commons licence, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line o he ma e ial. 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