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

Vol.: (0123456789) 1 3 Antonie van Leeuwenhoek (2023) 116:1–19 https://doi.org/10.1007/s10482-022-01778-w ORIGINAL PAPER FtsZ phosphorylation pleiotropically affects Z‑ladder formation, antibiotic production, andmorphogenesis inStreptomyces coelicolor PaulaYagüe · JoostWillemse · XianshaXiao · LeZhang · AngelManteca · GillesP.vanWezel Received: 3 January 2022 / Accepted: 27 August 2022 / Published online: 16 November 2022 © The Author(s) 2022 development, colony morphology, spore resistance, and antibiotic production in FtsZ knockout mutants expressing FtsZ alleles mimicking Ser319 and Ser387 phosphorylation and non-phosphorylation: AA (no phosphorylation), AE, EA (mixed), and EE (double phosphorylation). The FtsZ-eGFP AE, EA and EE alleles were not able to form observable FtsZ-eGFP ladders when they were expressed in the S. coelicolor wild-type strain, whereas the AA allele could form apparently normal eGFP Z-ladders. The FtsZ mutant expressing the FtsZ EE or EA or AE alleles is able to sporulate indicating that the mutant alleles are able to form functional Z-rings leading to sporulation when the wild-type FtsZ gene is absent. The four mutants were pleiotropically affected in colony morphogenesis, antibiotic production, substrate mycelium differentiation and sporulation (sporulation timing and spore resistance) which may be an indirect result of the effect in sporulation Z-ladder formation. Each mutant showed a distinctive phenotype in antibiotic production, single colony morphology, and sporulation (sporulation timing and spore resistance) indicating that the different FtsZ phosphomimetic alleles led to different phenotypes. Taken together, our data provide evidence for a pleiotropic effect of FtsZ phosphorylation in colony morphology, antibiotic production, and sporulation. Keywords Cell division· Differentiation· Serinephosphorylation· Sporulation· Streptomyces Abstract The GTPase FtsZ forms the cell division scaffold in bacteria, which mediates the recruitment of the other components of the divisome. Streptomycetes undergo two different forms of cell division. Septa without detectable peptidoglycan divide the highly compartmentalised young hyphae during early vegetative growth, and cross-walls are formed that dissect the hyphae into long multinucleoid compartments in the substrate mycelium, while ladders of septa are formed in the aerial hyphae that lead to chains of uninucleoid spores. In a previous study, we analysed the phosphoproteome of Streptomyces coelicolor and showed that FtsZ is phosphorylated at Ser 317 and Ser389. Substituting Ser–Ser for either Glu–Glu (mimicking phosphorylation) or Ala–Ala (mimicking non-phosphorylation) hinted at changes in antibiotic production. Here we analyse Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s1048202201778-w. P.Yagüe(*)· J.Willemse· X.Xiao· L.Zhang· G.P.vanWezel Department ofMolecular Biotechnology, Institute ofBiology Leiden, Leiden University, PO Box9505, 2300ABLeiden, TheNetherlands e-mail: [email protected] A.Manteca Departamento de Biología Funcional e IUOPA, Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, 33006Oviedo, Spain 2 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) Introduction Streptomycetes are filamentous bacteria with a complex multicellular lifecycle (Claessen et al. 2014). After germination, the hyphae grow out via tip growth and branching, forming an intricate network called the vegetative mycelium. This vegetative or substrate mycelium undergoes several rounds of programmed cell death, which is required for the formation of the reproductive aerial hyphae (Manteca etal. 2006; Yague etal. 2016). Eventually, the aerial hyphae differentiate into chains of spores (Flardh and Buttner 2009). The biochemical pathways regulating this complex way of growth have not yet been fully characterized (Claessen etal. 2006; McCormick and Flardh 2012). Streptomycetes produce a wide range of bioactive metabolites that are important for medicine, biotechnology, and agriculture (Barka et al. 2016; Berdy 2005; Katz and Baltz 2016). They are referred to as nature’s medicine makers (Hopwood 2007) and play a key role in the protection of eukaryotic hosts against challenges by pathogens (van Bergeijk etal. 2020). The production of secondary metabolites is closely linked to the developmental program of streptomycetes (van der Heul etal. 2018). Understanding the regulatory networks that control the physiology of this intriguing bacterium is one of the keys to understanding and improving secondary metabolism activation (Manteca and Yague 2018). Bacterial cell division has been studied extensively, whereby much information has been obtained particularly from the rod-shaped bacteria Escherichia coli or Bacillus subtilis (Du and Lutkenhaus 2017; Errington and Wu 2017). These bacteria grow by elongation of the lateral wall and divide by binary fission (Koch 2000). The process involves more than 35 proteins that form the so-called divisome at midcell. FtsZ is a tubulin-like GTPase protein (de Boer etal. 1992) that polymerizes into protofilaments that together form the Z-ring (Sun and Margolin 1998). The Z-ring is a cytoskeletal structure that forms the scaffold at the site of division and creates the constricting force that eventually divides the cell into two daughter cells (Vicente et al. 2006). The formation of the Z-ring is regulated negatively by the Min proteins that prevent division at the cell poles (Szwedziak and Ghosal 2017; Howard 2004), and nucleoid occlusion to prevent the formation of the septum over non-segregated chromosomes (Margolin 2004; Wu and Errington 2004). Various FtsZ-interacting proteins have been discovered that often interact with the conserved C-terminal end of the protein, showing that this is a hotspot for protein interactions (Huang etal. 2016). These interactions play a major role in the polymerization and correct localization of the Z-ring. The conserved C-terminal part (CCTP) of FtsZ interacts among others with the membrane tethers FtsA and ZipA (Hale and de Boer 1997; Pichoff and Lutkenhaus 2002), the Z-ring stabilising proteins ZapA (Low etal. 2004) and ZapD (Durand-Heredia etal. 2012), and with SepF, which is the only one of these proteins conserved in Streptomyces and forms ring-like structures that promote FtsZ polymerization (Hamoen etal. 2006; Krol etal. 2012; Singh etal. 2008). Conversely, the FtsZ-recruiting SsgB in Streptomyces interacts with the N-terminal part of FtsZ (Willemse et al. 2011). The precise FtsZ dynamics in bacterial cell division and the identity of the divisome proteins are still not fully understood (reviewed in (Barrows and Goley 2021). Cell division in the mycelial streptomycetes is coordinated differently, involving two types of cell division (Jakimowicz and van Wezel 2012). During vegetative growth, cell division results in cross-walls that divide the hyphae into long multinucleoid syncytial cells. Complex membrane assemblies thereby form chromosome-free zones in the hyphae during septum formation, apparently protecting the DNA from damage during division (Celler etal. 2016). During sporulation-specific cell division in the aerial hyphae, up to a hundred septa are laid down more or less simultaneously, eventually resulting in the formation of chains of uninucleoid spores (Jakimowicz and van Wezel 2012). Vegetative and sporulation-specific cell division also differ mechanistically, as illustrated by the fact that many cell division genes that are required for sporulation (e.g. ftsI, ftsL, and ftsW) are dispensable for vegetative cross-wall formation (Bennett etal. 2007, 2009; Mistry etal. 2008; Yague etal. 2016). Indeed, canonical cell division involving the divisome is only seen during sporulation. While in most bacteria cell division is negatively controlled, in streptomycetes FtsZ is actively recruited by the SsgB protein (Willemse etal. 2011). In turn, the localization of SsgB depends on its orthologue SsgA (Traag and van Wezel 2008), while SsgB is tethered to the membrane by SepG (Zhang etal. 2016). Thus the control of cell division differs substantially from rod-shaped 3 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) bacteria, most likely due to the absence of a midcell reference (Jakimowicz and van Wezel 2012). FtsZ is essential in most bacteria, but surprisingly, ftsZ mutants of Streptomyces are viable (McCormick etal. 1994; Dai and Lutkenhaus 1991). Dynamin-like proteins DynA and DynB were reported to stabilise Z-rings during Streptomyces sporulation (Schlimpert etal. 2017). Recent works discovered novel proteins associated with Streptomyces FtsZ during the vegetetative (Bush etal. 2022) and sporulation divisomes (Ramos-Leon etal. 2021). FtsZ is also involved in the formation of septa without detectable peptidoglycan in the highly compartmentalised young Streptomyces hyphae formed after spore germination and preceding the differentiation of the multinucleated substrate mycelium (Yague etal. 2016), making Streptomyces cell division even more complex. Besides the various layers of transcriptional regulation that control the major processes in cells, posttranslational modifications (PTMs) also play a major role. Protein phosphorylation is one of the most important PTMs in cells. His/Asp phosphorylation is a well-known PTM in prokaryotes, since it forms part of the two-component systems, while Ser/Thr/Tyr phosphorylation is one of the most important PTMs in eukaryotes (Petrickova and Petricek 2003). However, Ser/Thr/Tyr phosphorylation also exists in bacteria, where it has important regulatory roles, though it is still less well understood than in eukaryotes (Yague etal. 2019; Pereira etal. 2011; Petrickova and Petricek 2003). Members of the genus Streptomyces have one of the largest phosphoproteomes described to date (Parker etal. 2010; Manteca etal. 2011; Rioseras etal. 2018). The S. coelicolor genome encodes some 34 Serine Threonine Kinase proteins (STKs) and at least 184 phosphoproteins (Hempel etal. 2012; Parker et al. 2010; Rioseras etal. 2018; Petrickova and Petricek 2003; Manteca et al. 2011; Hirakata et al. 2019). To date, the biological relevance of Streptomyces Ser/Thr/Tyr protein phosphorylation was only experimentally validated for DivIVA, an essential protein that controls polar growth and hyphal branching (Hempel et al. 2012) and DnaA, also an essential protein controlling DNA replication (Lebkowski etal. 2020). DivIVA was also reported to be modulated by phosphorylation in Streptococcus suis (Ni etal. 2018). Important cellular bacterial processes were reported to be modulated by Serine/ Threonine/Tyrosine phosphorylation as cell-wall remodelling, quorum sensing or bacterial virulence [reviewed in Yague etal. (2019)]. The activity of FtsZ was reported to be modulated by S/T/Y phosphorylation in some bacteria as Deinococcus radiodurans (Maurya et al. 2018) and Mycobacterium tuberculosis (Thakur and Chakraborti 2006). Despite this knowledge, much more work will be necessary to fully characterise and understand the biological role of Streptomyces phosphoproteome and other bacterial phosphoproteomes. In a previous shotgun quantitative phosphoproteomic study, we discovered 131 phosphoproteins in S. coelicolor, one of which was FtsZ (Manteca etal. 2011; Rioseras etal. 2018). To investigate the importance and biological relevance of the FtsZ phosphorylations, we mutated residues Ser319 and Ser387 simultaneously, creating mutants mimicking FtsZ double phosphorylation (FtsZ-EE) and nonphosphorylation (FtsZ-AA). Preliminary analysis revealed that these mutations had an effect on secondary metabolism (Rioseras etal. 2018). In the current work, we further analyse the biological effect of FtsZ phosphorylation in the FtsZ-EE and FtsZ-AA mutants and in two new mutants mimicking single FtsZ phosphorylation at Ser319 or Ser387 (Mutants FtsZ-EA and FtsZ-AE) (Fig.1). We discovered that, in addition to secondary metabolism, FtsZ phosphorylation shows a surprising pleiotropic phenotype affecting Z-ladder formation during sporulation, colony morphogenesis, sporulation timing, spore morphology, and spore resistance. To the best of our knowledge, this is the first time that serine phosphorylation was described to interfere with FtsZ polymerisation and to affect biological processes different from secondary metabolism. Material andmethods Bacterial strains and media Streptomyces coelicolor A3(2) M145 was obtained from the John Innes Centre (UK) strain collection (Kieser 2000). Streptomyces coelicolor M145 was the parent for ftsZ* mutant strains, FtsZ-EE (pGlu319 and pGlu387; EE), FtsZ-AA (pGlu319 and Ala387; EA), FtsZ-EA (Ala387 and pGlu387; AE), and FtsZ-AE (Ala319 and Ala387; AA). Escherichia coli ET dam− 123,567 harbouring the 4 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) conjugative plasmid pUZ8002 (Paget et al. 1999) was used as host for conjugation. SFM (soya flavour, mannitol) agar medium (Hobbs 1989) was used as a sporulation medium and for the study of phenotypes using scanning electron microscopy, transmission electron microscopy or stereo microscopy. GYM medium (glucose 5 g/l, yeast extract 4g/l, malt extract 5g/l, 0.5g/l MgSO4·7H2O, agar 20g/l and supplemented to a final concentration of 0.5g/l K2HPO4) with cellophane disks was used as the growth medium for the confocal observations. MM (agar minimal medium) (Hopwood 1967), with mannitol as a carbon source, was used as a growth medium for checking the production of pigments (antibiotics) and the capacity of sporulation. For Streptomyces, agar plates were inoculated with 100ml of a suspension of 108 spores per ml, followed by incubation at 30°C. For E. coli, Lysogen broth [LB, (Bertani 2004)] liquid/solid medium supplemented with 20% of glucose was used followed by incubation at 37°C. E. coli strains harbouring the pBluescript II SK + and pNG3 plasmids were grown in ampicillin (100μg/ml) amended media. Nalidixic acid (25μg/ ml) was used in the E. coli/Streptomyces conjugation to inhibit E. coli (Kieser 2000). Streptomyces strains harbouring the integrative pNG3 plasmid (GonzalezQuinonez et al. 2016) were grown in SFM supplemented with apramycin (25 μg/ml) for sporulation. In order to prevent antibiotic interferences, the phenotypic analyses of the strains expressing the FtsZ alleles cloned into pNG3 were performed without antibiotic. Construction of ftsZ* mutant strains Different versions of recombinant ftsZ* whereby the codons for Ser319 and Ser387 were replaced by codons for either glutamate or alanine or combinations thereof were synthetized by GeneCust (www. genec ust. com) and cloned into pBluescript II SK + . For this, DNA fragments were amplified by PCR Fig. 1 Scheme of FtsZ and the two serine sites object of this study. A The amino acid sequence of FtsZ protein. The cartoon represents the FtsZ core structure (in red) from Mycobacterium tuberculosis (PDB ID 2Q1X). The linker region of Streptomyces coelicolor FtsZ spans residues 315–389 (in blue), while the C-terminal tail (CTT) spans residues 389–399 (in green), which are all depicted by balls. The side chain of Ser319 and Ser387 are labeled in red sticks. B FtsZ amino acid substitutions in the mutants. Substitutions of the 319 and 387 serine sites are highlighted in red. + (Glu) mimics permanent phosphorylation; − (Ala) mimics permanent lack of phosphorylation 5 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) from the S. coelicolor M145 chromosome using primers: FtsZ_F and FtsZ_R (Table 1). Integrative vector pNG3 (Gonzalez-Quinonez etal. 2016) or its derivatives pNG3-EE, -EA, -AE, and -AA harbouring a single copy of each recombinant ftsZ* gene (expressed from the native ftsZ promoter regions described in (Flardh etal. 2000) (Table1) were introduced into S. coelicolor M145 via conjugation using E. coli ET12567/pUZ8002 as a donor strain and following the protocol described in Kieser etal. (2000). This generated strains harbouring control plasmid or expressing FtsZ-EE, -EA, -AE, or -AA. Subsequently, the native ftsZ gene was inactivated using Crispr-Cas9 methodology in the four different transconjugants (FtsZ-EE, FtsZ-AA, FtsZ-EA, FtsZ-AE) eliminating the ftsZ ORF and generating the same ftsZ null background in all the phosphorylation/nonphosphorylation mimicking mutants (Tong et al. 2015). In the same way, a control strain harbouring ftsZ in pNG3 plasmid and subsequently inactivating the native copy, was performed demonstrating that the phenotypes of the mutants are not due to ftsZ placement (Supplementary Fig.1A). For Crispr-Cas9 the target sequence CGA TGA CTT TGA TGA CTG CG was used (provided by http:// staff. biosu stain. dtu. dk/ laeb/ crispy_ scoeli), the primer sgRNA-F (NcoI) and the primer sgRNA-R (SnaBI) (Table1). For the overlapping extremes (recombining fragment) the following primers were used to allow homologous region replacement, as described (Tong etal. 2015): 2082LeftF, 2082LeftR, 2082RightF, 2082RightR (Table 1). This resulted in the deletion of 1144 bp of ftsZ (nt positions 2,234,455–2,235,599) from the S. coelicolor genome (Tong etal. 2015). As a control, wild type strain in this study is harbouring empty plasmid pNG3 (Table1). Cell viability and morphology observations at the confocal microscope For the analysis of hypha viability, morphology and sporulation timing, cultures were grown on GYM, harvested at different time points from cellophanegrown mycelia, stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, L-13152) and observed under the confocal microscope following our previous protocol (Manteca etal. 2005). The LIVE/DEAD BacLight Bacterial Viability Kit consists in dry SYTO 9 and Propidium Iodide (PI) which we prepared at the concentrations recommended by Invitrogen, 6 µM and 30 µM respectively (both were prepared in ultrapure mQ water). Cellophane squares (1cm side) were manually cut, placed over the microscope slide, 20 µL of the SYTO9/PI mixture was added, a cover glass was carefully putted over the sample preventing bubble formation, the sample was incubated at room temperature for 5min, and immediately observed at the microscope. This kit uses SYTO9 and Propidium Iodide (PI), two DNA-binding colorants. SYTO9 penetrates intact membranes and stains viable cells green, whereas PI (staining red) only penetrates bacteria with damaged membranes. PI displaces SYTO9 from DNA when both colorants are present in dying cells. Samples were observed using an inverted Zeiss Axio Observer Z laser scanning microscope at wavelengths of 488 and 568nm for excitation and 530 (green) or 630 nm (red) for emission. For the analysis of septa formation, square microscopy cover glasses were positioned in SFM plates under an angle of 45°, and subsequently, 10μl of a spore suspension were inoculated (108 spores/ml). After 3–5days (depending on the strain) of incubation at 30°C, the cover glass was removed and 5µg/ ml of WGA-Alexa fluor 633 were added for cell-wall (peptidoglycan) staining. Samples were observed using an inverted Leica SP8 laser scanning microscope at wavelengths of 632 nm for excitation and 647nm for emission. Unstained samples (processed with ultrapure mQ water instead SYTO9/PI or WGA-Alexa fluor) were used as controls to fix the PMT gain levels at which autofluorescence was detected, which were much higher than the gain used to collect pictures. Autofluorescence, was extremely low, compared to the stained samples. At least three biological replicates were processed for each sample. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) was carried out as described (Colson etal. 2008). For this, small blocks of Streptomyces cultures grown on SFM medium were fixed using glutaraldehyde, dehydrated and dried. Eventually, the samples in 100% acetone were completely dried in a critical point dryer. Cells were mounted on an SEM stub and sputter-coated with platinum palladium to capture the images at 6 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) Table 1 Bacterial strains, plasmids and primers used in this study Description References Bacterial strains Escherichia coli TOP10 Harbouring the pBluescript II SK Invitrogen® Streptomyces coelicolor M145 SCP1-SCP2-harbouring empty plasmid pNG3 Kieser (2000) Streptomyces coelicolor M145 pGWS1574 ( habouring ftsZunder its own promoter on pNG3) This study E. coli ET12567/pUZ8002 E. coli ET12567 containing plasmid pUZ8002, a not self-transmissible plasmid which can mobilize other plasmids; ClnR, KmR Flett etal. (1997), MacNeil etal. (1992) Plasmids pNG3 Cloning vector; HygR, AmpRGonzalez-Quinonez etal. (2016) pBluescript II SK Cloning vector; AmpRAgilent® pCR™-Blunt II-TOPO® Zero Blunt®TOPO®PCR Cloning Kit, KmRInvitrogen® pHJL401 Cloning vector; TsrRLarson and Hershberger (1986) pCRISPR-Cas9 Conjugative and thermosensitive plasmid harbouring Cas9 Tong etal. (2015) pCRISPR-SgFtsZ pCRISPR-Cas9 harbouring the target SCO2082 sequence and a 2kb fragment surrounding the ftsZ ORF used to knockdown ftsZ This study FtsZ*-EE pNG3 pNG3 harboring ftsZ* pGlu319 and pGlu387; HygrR, AmpR, HygRRioseras etal. (2018) FtsZ*-AA pNG3 pNG3 harboring ftsZ* pGlu319 and Ala387; HygrR, AmpR, HygRRioseras etal. (2018) FtsZ*-EA pNG3 pNG3 harboring ftsZ* Ala387 and pGlu387; HygR, AmpR, HygRThis study FtsZ*-AE pNG3 pNG3 harboring ftsZ* Ala319 and Ala387; HygR, AmpR, HygRThis study FtsZ*-EE eGFP pHJL401 pHJL401 harbouring ftsZ*EE-egfp This study FtsZ*-AA eGFP pHJL401 pHJL401 harbouring ftsZ*AA-egfp This study FtsZ*-EA eGFP pHJL401 pHJL401 harbouring ftsZ*EA-egfp This study FtsZ*-AE eGFP pHJL401 pHJL401 harbouring ftsZ*AE-egfp This study Primers SCO4848F CGT CGT ATC CCC TCG GTT G Gonzalez-Quinonez etal. (2016) pMS82R GAG CCG GGA AAG CTC ATT CA Gonzalez-Quinonez etal. (2016) FtsZ_F GGA CTA GTA GCA GGG TGT GCG GAA G This study FtsZ_R AAG ATA TCC TAT CAC TTC AGG AAG TCCG This study sgRNA-F (NcoI) CAT GCC ATG GCG ATG ACT TTG ATG ACT GCG GTT TTA GAG CTA GAA ATA GC This study sgRNA-R (SnaBI) ACG CCT ACG TAA AAA AAG CAC CGA CTC GGT GCC This study 2082LeftF AGG CCT AGA CCG ACC ACC GCC GAG This study 2082LeftR CCT ATC ACT TCA GGA AGT CCG TGA TGA CTG CGA GGT AGT TCT G This study 2082RightF CAG AAC TAC CTC GCA GTC ATC ACG GAC TTC CTG AAG TGA TAG G This study 2082RightR AGG CCT AGT AAC CGA CCA CGG AAC GCA This study eGFP-FTSZ* F GTC AGA ATT CAG GCC TTC GAC GTG GCA GCA CCG CAG AAC TAC C This study eGFP-FTSZ* R GTC AAA GCT TGG ATC CTT CAG GAA GTC CGG CAC GTC C This Study 7 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) 15kV. SEM pictures were used for measuring spore length. The lengths of more than 250 spores were measured in each strain using the ROI manager plugin of ImageJ. Statistical significance was measured by comparing spore lengths in wild-type and the four mutants using a T-test. Three biological replicates; 3 plates/mutant, and three methodological replicates; 3 blocks per plate were used for SEM experiments. Transmission electron microscopy (TEM) For visualizing the spore chains of Streptomyces, small blocks were cut from SFM plates harvested in confluent cultures and processed for TEM essentially as described (Piette etal. 2005). The mycelium was washed with 1 × PBS before fixation with 1.5% glutaraldehyde, and blocks were then post-fixed with 1% osmium tetroxide for 30min. The samples were dehydrated by passing them through an ethanol gradient and placed in propylene oxide for 15min followed by incubation in a mixture of Epon and propylene oxide (1:1) and pure Epon (each step 45min). Finally, the samples were embedded in Epon and sectioned into 70nm slices, which were placed on 200-mesh copper grids. Samples were stained using uranyl-430 acetate (2%) and lead citrate (0.4%), if necessary, and imaged at 70kV in a Jeol 1010 transmission electron microscope. Three biological replicates; 3 plates/mutant, and three methodological replicates; 3 blocks per plate were used for SEM experiments. Stereo microscopy Single colonies of S. coelicolor and the mutant strains were observed using a Leica M80 stereomicroscope. Pictures were taken with a Leica DFC295 camera. Antibiotic measurements Undecylprodigiosin and actinorhodin were quantified spectrophotometrically according to Tsao etal. (1985) and Bystrykh etal. (1996). Cells were ruptured in the culture medium by adding 0.1N KOH. After vortexing and centrifugation, actinorhodin was quantified in the supernatant (ɛ640 = 25,320). Undecylprodigiosin was measured after vacuum drying of the mycelium, followed by extraction with methanol, acidification with HCl (to 0.5M), and a spectrophotometric assay (ɛ530 = 100,500). Reproducibility has been corroborated by at least three independent cultures. Resistance of spores to lysozyme, heating, and freezing For heating (55°C, 30min) and freezing (− 20°C, 24h) shock treatments, suspensions of 106 spores/ml were prepared in sterile distilled water and subjected to different treatments as detailed below. Germination of the spores before and after treatment was analysed by plating several dilutions and quantifying the number of colony-forming units (Rioseras etal. 2016). All quantifications were measured in triplicate. The data correspond to the average ± SD of the replicates. For lysozyme resistance, 106 spores/ml were plated in LB agar medium and directly placing filter discs containing 50μg, following the method of Kleinschnitz etal. (2011), and incubated at 30°C for 3days. ftsZ-eGFP alleles creation and observation at the confocal and fluorescence microscopes The pHJL401 plasmid (Larson and Hershberger 1986) (Table1), an E. coli/Streptomyces shuttle vector, was used for the constructions of the four different ftsZ* (Serine modifications) and the egfp expression under the control of the ftsZ promoter. The ftsZ promoter is cloned between sites EcoRI-StuI, the ftsZ* alleles without stop codon are cloned StuI-BamHI and the egfp is cloned downstream BamHI-NotI. The strains with the eGFP fusion were analyzed by Axio Observer Zeiss confocal microscope. Excitation was performed with a 488nm laser, and detection was performed with a 505–530 nm bandpass filter. The first 16h of growth, were observed making time-lapse experiments as follows; cultures were pre-grown on GYM medium for 6h at 30°C for the germination of the spores, samples were then excised out and inverted into µ-dishes (Ibidi GmbH 35mm, hight ibitreat). Pictures were taken every 10min during 16 h following Yagüe et al. (2016). Wild-type cultures (without FtsZ-eGFP fusions) were used as controls to fix the PMT gain levels at which autofluorescence was detected, which were much higher than the gain used to collect the pictures. For analysing the FtsZ-eGFP at later time points, cover glasses were positioned in SFM plates under an angle of 45°, and subsequently, 10μl of a spore suspension were 8 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) inoculated (108 spores/ml). At the indicated timepoints, cover glasses were removed, mounted with ultrapure mQ water, and observed using a Leica DMRXA fluorescence microscope with a FITC filter. Pictures were taken with an ORCA-Flash4.0 V3 Digital CMOS camera. Wild-type strain cultures (without FtsZ-eGFP fusions) were used controls to fix the levels at which autofluorescence was detected. At least three biological replicates were processed for each sample. Phase contrast images microscopy For analysing the sporulation of FtsZ*strains, cover glasses were positioned in SFM plates under an angle of 45°, and subsequently, 10 μl of a spore suspension were inoculated (108 spores/ml). After 5–7days, depending on the strain, cover glasses were removed, mounted with ultrapure mQ water, and observed using phase contrast under the Leica DMRXA microscope. Pictures were taken with an ORCA-Flash4.0 V3 Digital CMOS camera. Image processing Microscopy images were processed (histogram intensity levels were adjusted and scale added) using the Fiji software (Schindelin et al. 2012). Figure composites were made using AdobePhotosop CS5.1 and Adobe Photoshop 2021. Results Construction of Streptomyces coelicolor strains harbouring ftsZ* alleles mimicking FtsZ phosphorylation/non-phosphorylation Streptomyces coelicolor FtsZ (SCO2082) is differentially phosphorylated at Ser319 and Ser387 during the cell cycle (Rioseras etal. 2018; Manteca etal. 2011). These serine residues are located in the protein linker, just between the global domain and the C-terminal part of FtsZ (Fig.1A). With the aim of analysing the effect of FtsZ serine-phosphorylation in S. coelicolor, we used a well-established methodology based on the substitution of the Ser residues by Glu or Ala, thus mimicking phosphorylation or non-phosphorylation, respectively (Zhao et al. 1994; Keller-Pinter et al. 2017; Hewitt etal. 2017; Morrison etal. 2003; Trutnyeva et al. 2005). Four different mutant variants were studied: two mutants previously created, namely FtsZ-EE (EE; mimicking two phosphorylations) and FtsZ-AA (AA; mimicking non-phosphorylation) (Rioseras et al. 2018); and two new mutants mimicking phosphorylation in only one of the serines, namely FtsZ-EA Glu-Ala (EA; mimicking phosphorylation at Ser319 and no phosphorylation at Ser387), and FtsZ-AE Ala-Glu (AE; mimicking no phosphorylation at Ser319 and phosphorylation at Ser387) (Fig. 1B). The strains, each expressing one of the FtsZ variants, were generated by the introduction of the mutant copies into an ftsZ null background. For this, the recombinant ftsZ*—transcribed from the native ftsZ promoter region (Flardh etal. 2000)—was introduced into integrative vector pNG3 (GonzalezQuinonez etal. 2016) and introduced into S. coelicolor M145. The native ftsZ was subsequently inactivated using Crispr-Cas9. As detailed below, each mutant showed a distinctive phenotype in antibiotic production, single colony morphology and sporulation (sporulation timing and spore resistance) indicating that the different FtsZ phosphomimetic alleles led to different phenotypes. Phosphorylation affects FtsZ Z-ladder formation during sporulation. To study Z-ring and Z-ladder formation (1µm spaced Z-rings), we followed the methodology developed by Grantcharova etal. (2005), where ftsZ-eGFP is ectopically expressed in a S. coelicolor strain carrying the native ftsZ gene. This approach was demonstrated to be useful to form functional FtsZ-eGFP Z-rings that can be observed during development and was largely replicated in Streptomyces [see for instance Willemse and van Wezel (2009), Yague etal. (2016), Bush etal. (2022)].We expressed FtsZ-eGFP fusions from the native ftsZ promoter region as previously described (Grantcharova etal. 2005). This was done for all four mutants (expressing FtsZ-AA, AE, EA and EE) and for wild-type fstZ. The constructs were introduced into the S. coelicolor M145 and Z-ring formation was analysed using fluorescence microscopy (Fig.2). During Streptomyces development, there are two stages showing massive Z-ring formation. The first one during the early development (the first 16h culture) corresponding to the early compartmentalised mycelium 9 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol.: (0123456789) Fig. 2 Z-rings and ladders formation in the ftsZ*- eGFP alleles. Fluorescence micrographs of the young compartmentalised hyphae (growing on GYM), substrate and aerial mycelium (growing on SFM) and sporulating hyphae (growing on SFM) are shown. Contrast mode images are shown for the FtsZ-EE, FtsZ-EA, and FtsZ-AE mutants to illustrate that they are sporulating. Only FtsZ-AA form sporulation Z-ladders. Representative images from at least three biological replicates are shown. Arrows indicate Z-rings. Arroheads indicate FtsZ-eGFP accumulations that do not cross the transverse axis of the hyphae and do not constitute mature Z-rings. Scale bars 10µm 16 Antonie van Leeuwenhoek (2023) 116:1–19 1 3 Vol:. (1234567890) Author contributions PY, JW, and XX performed the experiments. PY made the figures. PY, AM, and GPVW planned the experiments and wrote the manuscript. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. PY was funded by a Marie-Curie-Clarín cofund fellowship (FICYT, “Consejería de Educación y Ciencia, Principado de Asturias, Spain”). Work in AM’s lab was funded by “Ministerio de Ciencia, Innovación Universidades/Agencia Estatal de Investigación/Fondo Europeo de Desarrollo Regional” (Project RTI2018-093978-B-I00), and the “Consejería de Empleo, Industria y Turismo del Principado de Asturias” (Project FC-GRUPIN-IDI/2018/000120). Declarations Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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