The Role of SepF in Cell Division and Diazotrophic Growth in the Multicellular Cyanobacterium Anabaena sp. Strain PCC 7120

Microbiological Research 277 (2023) 127489 Available online 12 September 2023 0944-5013/© 2023 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The role of SepF in cell division and diazotrophic growth in the multicellular cyanobacterium Anabaena sp. strain PCC 7120 A. Valladares , S. Picossi , L. Corrales-Guerrero 1 , A. Herrero * Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Seville, Spain ARTICLE INFO Keywords: Cell division Heterocyst-forming cyanobacteria SepF-FtsZ interactions Septal junctions ABSTRACT The cyanobacterium Anabaena forms filaments of cells that grow by intercalary cell division producing adjoined daughter cells connected by septal junction protein complexes that provide filament cohesion and intercellular communication, representing a genuine case of bacterial multicellularity. In spite of their diderm character, cyanobacterial genomes encode homologs of SepF, a protein normally found in Gram-positive bacteria. In Anabaena, SepF is an essential protein that localized to the cell division ring and the intercellular septa. Overexpression of sepF had detrimental effects on growth, provoking conspicuous alterations in cell morphology that resemble the phenotype of mutants impaired in cell division, and altered the localization of the division-ring. SepF interacted with FtsZ and with the essential FtsZ tether ZipN. Whereas SepF from unicellular bacteria generally induces the bundling of FtsZ filaments, Anabaena SepF inhibited FtsZ bundling, reducing the thickness of the toroidal aggregates formed by FtsZ alone and eventually preventing FtsZ polymerization. Thus, in Anabaena SepF appears to have an essential role in cell division by limiting the polymerization of FtsZ to allow the correct formation and localization of the Z-ring. Expression of sepF is downregulated during heterocyst differentiation, likely contributing to the inhibition of Z-ring formation in heterocysts. Finally, the localization of SepF in intercellular septa and its interaction with the septal-junction related proteins SepJ and SepI suggest a role of SepF in the formation or stability of the septal complexes that mediate cell-cell adhesion and communication, processes that are key for the multicellular behavior of Anabaena. 1. Introduction In the vast majority of bacteria, the essential process of cell division is orchestrated by the so-called Z-ring, the complex of a few proteins that includes the tubulin structural homolog FtsZ and a few others that link FtsZ to the cytoplasmic membrane and regulates FtsZ polymerization to constitute a discontinuous ring encircling the cell cytoplasm at midcell. The Z-ring recruits and localize further proteins to assemble the divisome that directs the synthesis of the polar peptidoglycan caps of the daughter cells and, finally, envelope fission and daughter cell separation (see e.g., Du and Lutkenhaus, 2017). Although FtsZ is ubiquitous in bacteria, a diversity is found in other components of the Z-ring, including FtsZ tethers to the cytoplasmic membrane, as well as in divisome components. Variations have also been found in mechanisms of spatial regulation of the divisome and cell division progress, responding, among other factors, to the bacterial morphology or life style. Widespread FtsZ membrane tethers, which also impact the polymerization of FtsZ, are the proteins FtsA or ZipA, which furthermore contribute to stabilize the Z-ring and are essential proteins in Escherichia coli. FtsA has a C-terminal amphipathic helix for localization to the membrane (Pichoff and Lutkenhaus, 2005), whereas ZipA has an N-terminal transmembrane helix (Hale and de Boer, 1997). Both FtsA and ZipA bind to the C-terminal peptide of FtsZ (Mosyak et al., 2000; Szwedziak et al., 2012; Du et al., 2015). Gram-positive bacteria also bear the protein EzrA, which presents an N-terminal transmembrane helix and a C-terminal FtsZ-binding domain and, hence, could provide membrane anchoring to FtsZ (Singh et al., 2007). EzrA is essential in the ovococcus Streptococcus pneumoniae (P´ erez et al., 2021). In Bacillus subtilis neither FtsA nor EzrA are essential, and both proteins can be deleted together, although division defects are observed when both are absent (Duman et al., 2013). The ylmF gene (sepF) was identified in S. pneumoniae as a gene downstream from ftsA and ftsZ in the ylm cluster, whose inactivation resulted in abnormal cell morphology and septation (Fadda et al., 2003). * Corresponding author. E-mail address: [email protected] (A. Herrero). 1 Present address: Instituto de Investigaciones Químicas, CSIC and Universidad de Sevilla, Seville, Spain. Contents lists available at ScienceDirect Microbiological Research journal homepage: www.elsevier.com/locate/micres https://doi.org/10.1016/j.micres.2023.127489 Received 20 July 2023; Received in revised form 23 August 2023; Accepted 9 September 2023 Microbiological Research 277 (2023) 127489 2 Later, YlmF was identified as a new component of the B. subtilis Z-ring that interacts with FtsZ and is recruited to the division site in an FtsZ-dependent manner (Ishikawa et al., 2006; Hamoen et al., 2006). Indeed, SepF binding to the C-terminal tail of FtsZ was shown in vitro (Singh et al., 2008). Although disruption of ylmF alone had minor effects, the mutation was synthetically lethal in combination to ftsA inactivation, whereas the effects of ftsA inactivation could be complemented by ylmF overexpression (Ishikawa et al., 2006). Deletion of sepF was also lethal in combination with ezrA deletion (Hamoen et al., 2006). The structure of SepF includes a globular C-terminal domain, which contains the determinants for FtsZ binding, and an N-terminal amphipathic helix that provides a membrane anchor (Duman et al., 2013). SepF dimerizes and forms curved polymers at the leading end of the division septa, on top of which FtsZ filaments would assemble determining septal thickness (Duman et al., 2013; Wenzel et al., 2021). Indeed, a structure of the C-terminal core of SepF from Corynebacterium glutamicum in complex with the C-terminal domain of FtsZ has been reported (Sogues et al., 2020). Additionally, complexes of FtsZ and its interacting proteins, including SepF and EzrA, have been recently imaged in living B. subtilis cells, showing that these proteins affect FtsZ filament condensation to form the fully functional Z-ring ultimately necessary for cytokinesis (Squyres et al., 2021). In Actinobacteria, which lack FtsA and ZipA, SepF is essential and midcell septa are not formed in its absence (Sogues et al., 2020; Gola et al., 2015; Gupta et al., 2015), except in the genus Streptomyces, possibly due to the presence of SepF homologs (see White and Eswara, 2021). In M. tuberculosis, in addition to interacting with FtsZ, SepF also interacts with MurG, which is involved in the synthesis of the peptidoglycan precursor lipid II, suggesting a link between cell division and peptidoglycan synthesis (Gupta et al., 2015), as reported earlier for B. subtilis (Hamoen et al., 2006). Thus, SepF is widespread in Gram-positive bacteria and its role has been well studied in some model strains such as B. subtilis. However, much less is known about its role in other bacteria with disparate physiological features and even distinctive mechanisms in the process of cell division (see White and Eswara, 2021). Cyanobacteria are phototrophs whose metabolism is mainly oriented to the assimilation of inorganic carbon and nitrogen through oxygenic photosynthesis. They show a large morphological diversity ranging from unicellular to complex, including filamentous forms that represent genuine examples of bacterial multicellularity. This is the case of the model strain Anabaena sp. strain PCC 7120 (hereafter Anabaena), in which the organismic unit is a filament of communicated cells (Herrero et al., 2016). Anabaena cells have an outer membrane and, hence, it is considered a Gram-negative bacterium. However, although the cytoplasmic membrane is individual for each cell, the outer membrane is continuous along the filament, delimiting a continuous periplasmic space that is shared by all the cells in the filament (see Herrero et al., 2016). Moreover, depending on the environmental conditions, the filament can include two cell types specialized in different functions. Thus, when combined nitrogen is available, the filament includes only one type of cells. In contrast, when filaments are transferred to medium lacking combined nitrogen, heterocysts, which are cells specialized for the fixation of atmospheric nitrogen, differentiate at semiregular intervals along the filament (Flores and Herrero, 2010; Zeng and Zhang, 2022). Heterocyst differentiation is a tightly regulated process, which includes activation of multiple genes and repression of others, leading to lack of oxygenic photosynthesis activity and expression of nitrogenase activity (Flores et al., 2019). The Anabaena filament grows by intercalary cell division involving specific mechanisms to produce adjoined daughter cells instead of separated daughters as found in most bacteria (see Herrero et al., 2016). The cells in the filament are moreover connected by proteinaceous septal structures that provide cell-cell cohesion and intercellular communication involving molecular exchanges (see Flores et al., 2016). Regarding Z-ring components, Anabaena possess an FtsZ protein that, in addition to the widespread domains found in the orthologs from common bacterial models, includes an essential N-terminal peptide of ca. 60 residues that is required for proper FtsZ polymerization and cell division (Corrales-Guerrero et al., 2018). This N-terminal peptide of FtsZ is characteristic of filamentous cyanobacteria capable of cell differentiation. Cyanobacteria in general possess a distinct FtsZ-interacting protein, ZipN (Koksharova and Wolk, 2002; see Cassier-Chauvat and Chauvat, 2014), which in Anabaena is essential and represents a main FtsZ membrane tether (Camargo et al., 2019). Notably, in spite of its Gram-negative character, many cyanobacteria, including Anabaena, also possess putative sepF homologs (Cassier-Chauvat and Chauvat, 2014). SepF has been studied in two unicellular cyanobacteria: the cylindrical Synechococcus sp. strain PCC 7942, in which sepF is essential and its depletion leads to the formation of long cells (Miyagishima et al., 2005), and the spherical Synechocystis sp. strain PCC 6803, in which depletion of SepF, which is also essential, leads to the formation of giant cells (Marbouty et al., 2009). Here, we have addressed the study of SepF in Anabaena, including its spatiotemporal regulation of expression along the filament, its role in growth and heterocyst differentiation, its interactome and its effects on the in vitro polymerization of FtsZ. We provide evidence for an essential role of SepF in cell division in this cyanobacterium. 2. Materials and methods 2.1. Strains and growth conditions Anabaena sp. strain PCC 7120 and derived mutant strains were grown in BG11 medium (containing NaNO 3 as a nitrogen source) (Rippka et al., 1979). For incubation with other nitrogen sources, BG11 0 (containing no combined nitrogen) and BG11 0 plus ammonium (4–6 mM NH 4 Cl and 8–12 mM TES-NaOH buffer, pH 7.5, instead of NaNO 3 ) media were used. Cultures were incubated at 30 ◦C with illumination (12 μ E m −2 s −1 white light emitted from Osram LED lamps 16.4 W/4000 K) in Erlenmeyer flasks with shaking or in plates of medium solidified with 1% Difco agar. For the mutants, media were supplemented with antibiotics: neomycin sulphate (Nm) at 25 μ g ml −1 in solid medium or 5 μ g ml −1 in liquid medium (CSAV71, CSAV54 and CSAV73); spectinomycin dihydrochloride pentahydrate (Sp) and streptomycin sulphate (Sm) were added at 5 μ g ml −1 each in solid medium or 2.5 μ g ml −1 each in liquid medium (CSAV52, CSAV53, CSAV54) or at 6 μ g ml −1 in solid or liquid medium (CSAV64, CSAV73). The growth of cultures was monitored by measuring the absorbance at 750 nm in aliquots withdrawn at the indicated times. The chlorophyll (Chl) content of the cultures was determined after extraction with methanol (Mackinney, 1941). In Anabaena, 1 μ g Chl corresponds to ca. 3.3 ×10 6 cells. For the calculation of growth rates, filaments of the mutants grown in the presence of antibiotics were transferred to media without antibiotics. (Table S1 in the Supplementary material lists all used cyanobacterial strains and plasmids.). 2.2. Plasmid and strain constructions Strain CSAV52 contains a sfgfp-sepF fusion (encoding sfGFP-4Gly- SepF) expressed from the P sepF promoter, together with a native copy of P sepF -sepF. To generate it, two DNA fragments, one containing the sepF promoter sequence and another containing the sepF coding region were amplified by PCR using Anabaena genomic DNA as template and primer pairs alr0487–27/alr0487–28 and alr0487–31/alr0487–32, respectively (all oligodeoxynucleotide primers used are listed in Table S2 in the Supplementary material). Also, a fragment containing the sfgfp gene was amplified using plasmid pCSAL39 (Corrales-Guerrero et al., 2014) and primers alr0487sf-29/alr0487sf-30 (alr0487sf-30 encodes 4 Gly residues). These fragments were joined together by the use of overlapping PCR with primers alr0487–27/alr0487–32. The resulting KpnI-ended fragment was cloned into KpnI-digested pCSV3 vector (Valladares et al., 2011) producing plasmid pCSAV325 (all plasmids generated in this A. Valladares et al. Microbiological Research 277 (2023) 127489 3 work were verified by sequencing), which was transferred to Anabaena by conjugation (Elhai et al., 1997) with selection for Sm and Sp (resistance is encoded in the vector portion of the transferred plasmid). One clone that had incorporated the transferred plasmid by a single cross-over event in the sepF genomic region was selected and named strain CSAV52. Strain CSAV53 expresses a gfp-mut2 gene from the sepF gene promoter. To generate it, a DNA fragment of 436 bp encompassing the first six codons and sequences upstream of sepF was amplified by PCR using Anabaena DNA as the template and primer pair alr0487–22/alr0487–23 and cloned into plasmid pCSEL22 (Olmedo-Verd et al., 2006) rendering plasmid pCSAV304, which includes a fusion of the sepF promoter plus the sequences encoding the first six SepF residues and the promoter-less gfp-mut2. Plasmid pCSAV304 was transferred by conjugation with selection for Sm and Sp (resistance is encoded in the vector portion of the transferred plasmid). One clone that had incorporated the transferred plasmid by a single cross-over event in the sepF genomic region was selected and named strain CSAV53. Strain CSAV54 carries the replicative plasmid pCSAV308 encoding a copy of the sepF gene expressed from the nitrogen regulated P ND promoter. To generate it, two DNA fragments of the sepF region were amplified by PCR: fragment 1 comprises sequences upstream of sepF (amplified with primers alr0486–6/alr0487–4); fragment 2 comprises sequences of sepF and part of the downstream ORF alr0488 (amplified with primers alr0487–5/alr0488–5). Both fragments were inserted into plasmid pCSFR15 (Camargo et al., 2019), which includes the P ND promoter (Ramos-Le´ on et al., 2015) preceded by gene-cassette C.S3 encoding resistance to Sm and Sp (Elhai and Wolk, 1988), generating plasmid pCSL135. In this plasmid, fragment 1 precedes the C.S3 sequence, and fragment 2 follows the P ND sequence. The insert of pCSL135 was transferred to the replicative vector pRL200 (pRL25C [Wolk et al., 1988] with an extended polylinker; CP Wolk, unpublished), which includes a Nm R determinant, generating plasmid pCSAV308, which was transferred by conjugation to Anabaena with selection for Nm, Sm and Sp. One exconjugant was selected and named strain CSAV54. Strain CSAV64 includes the gene construct P trc -sepF. A 474-bp DNA fragment (encoding from the start codon to Ser157) of the sepF gene was amplified by PCR with genomic Anabaena DNA and primers P trc SepF-1/ P trc SepF-2 and cloned, in frame with the site of initiation of translation located after the trc promoter, into the expression vector pTrc99A (Amann et al., 1988). Afterwards, the C.S3 gene cassette was introduced into the lacI q gene in the pTrc99A portion, rendering plasmid pCSAV328. To avoid deleterious effects of constitutive expression of sepF, plasmid pCSAV328 was maintained in E. coli BL21 (DE3) (Studier et al., 1990) in the presence of pREP4 (Qiagen), which expresses high levels of the LacI q repressor. Plasmid pCSAV328 was transferred to Anabaena by conjugation with selection for Sm and Sp. One clone that had integrated pCSAV328 in the sepF genomic region was selected and named strain CSAV64. Strains CSAV71 and CSAV73 express a sfgfp-zipN fusion gene, besides the native zipN, both from the zipN promoter in the zipN genomic locus. To generate them, plasmid pCSCV37 (Vel´ azquez-Su´ arez et al., 2022) was transferred to the WT (with selection for Nm R ) and the CSAV64 strain (with selection for Sm R , Sp R , Nm R ), generating strains CSAV71 and CSAV73, respectively. One clone resulting from each conjugation that had inserted the transferred plasmid by a single crossover into the zipN locus leaving an intact P zipN -zipN gene was selected. To express in E. coli a version of the Anabaena sepF gene preceded by a sequence encoding a 6His-tag, two DNA fragments were amplified by PCR. Fragment 1 comprises the 6His-tag sequence together with the site for rTEV protease digestion. This fragment was generated using vector PROEX-HTb (Invitrogen) as the template and primers pProEX-NcoI/ alr0487–36. Fragment 2 comprises the sepF coding region amplified by PCR using Anabaena DNA as the template and primers alr0487–35/ alr0487–33. Fragments 1 and 2 were joined together by overlapping PCR with primers pProEX-NcoI/alr0487–33. The resulting NcoI XhoI- ended fragment was cloned into NcoI XhoI -digested pET28b vector producing plasmid pCSAV339, which was transferred to E. coli strain BL21. To express in E. coli a version of the Anabaena sepF gene preceded by a sequence encoding a Strep-tag II, a DNA fragment was amplified by PCR using Anabaena DNA as the template and primers alr0487–6/ alr0487–7. The product obtained was cloned in vector pCSMN28b, which is based on pET28b but carries a Strep-tag II-encoding sequence instead of the 6 His-encoding sequence (Napolitano et al., 2013), producing plasmid pCSL141b. The NcoI XhoI fragment from pCSL141b plasmid was then cloned in pCOLADuet-1 (Novagen®) vector digested with the same enzymes, producing pCSL141, which was transferred to E. coli strain BL21. 2.3. FtsZ and SepF purification and in vitro polymerization FtsZ was purified as previously described (Corrales-Guerrero et al., 2018). Strep-tag II-SepF was purified from E. coli (pCSL141) (see above). For preparation of cell extracts, cultures were supplemented with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and incubated for 3 h at 37 ◦C. Then, the cells were harvested by centrifugation for 10 min at 5, 000 rpm. The cell pellet was washed with buffer A (50 mM Tris-HCl [pH 7.5], 300 mM KCl, 10% glycerol) and resuspended in ice-cold buffer A (5 ml g −1 of cells) containing protease inhibitor cocktail complete Mini EDTA-free (Roche) and homogenized on ice for 5 min. The resulting cell suspension was incubated with 1 mg ml −1 lysozyme for 1 h, at 4 ◦C and then subjected to six pulses, 30 s each, of ultrasonication. The lysate was cleared by centrifugation at 15000 g for 30 min at 4 ◦C. The soluble fraction was passed through StrepTrap HP columns using as eluent desthiobiotin (GE Healthcare) (0.53 mg ml –1 in the same buffer). The eluted fractions were subjected to filtration through GE Healthcare PD-10 columns (Sephadex G-25 M), and Strep-tag II-SepF protein was finally dissolved in a buffer containing 50 mM HEPES (pH 7.2), 50 mM KCl, 5% glycerol. 6His-SepF was purified from E. coli bearing plasmid pCSAV339 (see above). After induction with 1 mM IPTG and preparation of cell lysates as above, SepF was purified by passing through 1 ml His-select columns (GE Healthcare) using imidazole to elute the retained proteins. After filtration through PD-10 columns, His-SepF was finally dissolved in buffer containing 50 mM HEPES (pH 7.2), 50 mM KCl, 5% glycerol. For sedimentation assays, two samples of His-FtsZ (1.2 μ M) were incubated alone for 20 min at 30ºC in 50 mM HEPES (pH 7.2), 50 mM KCl, 10 mM MgCl 2 and 5% glycerol, supplemented with 2 mM GTP; then His-SepF (7 μ M) was added to one of the samples and proteins were incubated for further 30 min. After centrifugation at 150,234 g for 15 min, aliquots of the supernatant and sediment fractions were run on 10% SDS-PAGE gels, and the protein bands scanned and quantified using ImageJ 1.47i software. For visualization of FtsZ polymers by TEM, purified FtsZ protein was incubated at 30ºC in the presence or absence of Strep-tag II-SepF, as indicated, in the same buffer used for sedimentation assays. 2.4. Analysis of sepF expression by northern blot For RNA isolation, Anabaena filaments growing exponentially (3 μ g Chl/ml) in BG11 0 C medium (BG11 0 medium supplemented with 10 mM NaHCO 3 ) plus ammonium and bubbled with a mixture of CO 2 (1% [vol/ vol]) and air, were harvested at room temperature and either used directly or washed with BG11 0 C medium, resuspended in BG11 0 C medium at the same cell density and further incubated under culture conditions for the indicated periods of time. Total RNA and northern blot analysis were performed as described (Paz-Yepes et al., 2007) with 7 μ g RNA loaded per lane. The sepF probe was an internal DNA fragment amplified by PCR using Anabaena genomic DNA as template and primer pair alr0487–8/alr0487–9. A probe of the rpnB gene amplified from A. Valladares et al. Microbiological Research 277 (2023) 127489 4 plasmid pT7–7120 (Vioque, 1997) with the Universal and Reverse primers was used for normalization. Radioactive areas in membranes were visualized and quantified with a Cyclone storage phosphor system (Packard). 2.5. BACTH analysis BACTH assays were based on the reconstitution of adenylate cyclase from Bordetella pertussis (Karimova et al., 2005). Fusions of the genes encoding the adenylate cyclase T18 and T25 fragments to the genes ftsZ, zipN, sepF, zipS, ftsE, ftsX, ftsI, ftsQ, ftsW and sepJ were as previously described (Ramos-Le´ on et al., 2015; Corrales-Guerrero et al., 2018; Camargo et al., 2019). Plasmids were transformed into E. coli XL1-Blue for amplification. Isolated plasmids were co-transformed into strain BTH101 (cya-99), and the resulting clones were plated on a solid LB medium containing selective antibiotics and 1% glucose. Co-transformants were grown in a liquid medium in the presence of IPTG plus antibiotics and supplemented with o-nitro- phenol-β-galactoside. The o-nitrophenol (ONP) produced per mg of protein versus time was represented, and the β-galactosidase activity was deduced from the slope of the linear function. 2.6. Microscopy GFP fluorescence was monitored with an Olympus FLUOVIEW FV3000 (hyper-resolution) confocal laser-scanning microscope equipped with an UPlanApo 60 ×1.5 NA oil immersion objective (excitation, 488-nm; collection 500–540 nm for GFP or excitation, 640 nm; collection, 650–750 nm for cyanobacterial autofluorescence). For observation of FtsZ filaments, aliquots of the preparations were blotted dry into carbon-coated copper grids, stained with 1% uranyl acetate and observed and photographed with a Libra 120 (Zeiss) transmission electron microscope, essentially as described (Corrales-Guerrero et al., 2018). 3. Results 3.1. Spatiotemporal regulation of sepF expression in Anabaena In contrast to many Gram-positive bacteria (see White and Eswara, 2021), in Anabaena, the ylm gene cluster is not linked to the gene ftsZ, but instead is found in a different chromosomal location, downstream from an operon including the pipX gene, which is involved in transcriptional regulation of late heterocyst differentiation genes, and ORF alr0486 (Fig. 1A). In this cluster, alr0487 encodes a SepF homolog, alr0488 is a putative proC gene (encoding pyrroline carboxylate reductase), and alr0489 and alr0490 encode hypothetical proteins. We had previous evidence of co-transcription of sepF with the downstream ORFs alr0488 and alr0489, which however is independent of transcription of the upstream operon pipX-alr0486 (Valladares et al., 2011). Here, we performed northern blot analysis with RNA extracted from filaments grown with ammonium and incubated for 24 h in the absence of combined nitrogen and a probe internal to alr0487. A strong hybridization signal, corresponding to a transcript of ca. 0.6 kb (alr0487 is 597 bp long), was detected in both conditions, and much weaker signals corresponding to longer transcripts were detected as well (Fig. 1B). Thus, a strong polarity in the sepF operon appears to take place, with the sepF message being much represented than those of the downstream ORFs alr0488 and alr0489. Higher expression of alr0487 than of alr0488 and alr0489 was also evident in a global transcriptomic study of Anabaena (Flaherty et al., 2011). Additionally, an intergenic transcription start site has been detected between alr0487 and alr0488 (Mitschke et al., 2011), indicating that alr0488 could be transcribed independently of sepF. Finally, transcript levels of sepF increased after N-stepdown; e.g., 1.9-fold after 24 h of incubation in the representative experiment shown in Fig. 1C. These results are in agreement with data from (Flaherty et al., 2011) that showed an increase of 2.8 times in the alr0487 transcript level 12 h after N-stepdown and of 1.8 times after 21 h (see also Camargo et al., 2021). To study the spatiotemporal pattern of expression of sepF in the presence of combined nitrogen and during heterocyst differentiation, we generated strain CSAV53, which bears a fusion of the sepF promoter to the gfp-mut2 gene in the native sepF locus, keeping an intact copy of sepF also expressed from its native promoter (Fig. 1D). Fluorescence from GFP was monitored in filaments of CSAV53 grown with ammonium and at different times after the transfer to combined nitrogen-free medium (Fig. 1E). Under our experimental conditions, heterocyst differentiation took about 18–24 h to complete. In the presence of ammonium, GFP fluorescence was detected in all the cells of the filament. After N-step- down, fluorescence was observed to diminish in the cells that were differentiating into heterocysts (which can be recognized by loss of cyanobacterial autofluorescence and changes in cell morphology). Loss of GFP fluorescence was an early event that could be already detected 3 h after the transfer, when morphological differentiation was still hardly detectable. GFP fluorescence became undetectable in immature and mature heterocysts, the latter recognized by their polar refringent granules (see 18, 24 and 30 h in Fig. 1E). In vegetative cells, fluorescence intensity increased upon N-stepdown (1.4 times after 24 h) (Fig. 1E). Notably, a first peak of fluorescence was detected after ca. 3 h, which is consistent with data on northern blot analysis shown above. 3.2. Localization of SepF in Anabaena The Anabaena SepF protein would have 198 amino acid residues, exhibiting 35% identity to B. subtilis SepF, and higher identity in its C- terminal half, which, as mentioned above, contains the determinants for binding to FtsZ. As in the case of B. subtilis (Duman et al., 2013) and C. glutamicum (Sogues et al., 2020) SepF, the Anabaena protein is predicted to include an amphipathic helix membrane anchor in its N-ter- minal end (predicted with Amphipaseek [Sapay et al., 2006]). To study the localization of SepF in the Anabaena filaments, we generated strain CSAV52 that bears a sfgfp-sepF gene encoding a sfGFP fused to the N-terminus of SepF, expressed from the sepF promoter, besides an intact copy of sepF (Fig. 2A). In filaments of strain CSAV52 grown with combined nitrogen, either ammonium or nitrate, many cells exhibited GFP fluorescence in bands localized at midcell and also at the intercellular septal regions in recently divided cells (Fig. 2B, C). Considerable fluorescence dispersed in the cell cytoplasm was also observed, which can result from the fact that amphipathic helices provide relatively weak and reversible interaction with membranes, as previously considered (Saaki et al., 2022). Also, the N-terminal GFP fusion might to some extent interfere with SepF localization to membrane. Upon N-stepdown, midcell and, occasionally, septal fluorescence bands could be detected in vegetative cells, but never in the cells differentiating into heterocysts or in mature heterocysts (Fig. 2B, C). Dispersed fluorescence also decreased during differentiation, so that it became low in mature heterocysts (recognized by polar refringent granules), consistent with lack of sepF expression in heterocysts as described above. In summary, SepF is localized to the divisome and the recently-formed septa, and upon N-stepdown the expression of the sepF gene ceases in the differentiating cells and increases in the vegetative cells. 3.3. Effects of alterations of sepF expression in Anabaena To gain insight into specific roles of SepF in Anabaena, we attempted the generation of mutant strains lacking an intact sepF gene. However, different procedures that included the transfer to Anabaena of constructs including sepF versions with internal in frame deletions to substitute for the native P sepF -sepF locus failed to yield segregation of the altered gene allele. We then generated strains bearing extra copies of sepF. Strain CSAV54 carries a replicative plasmid encoding the native sepF gene A. Valladares et al. Microbiological Research 277 (2023) 127489 5 Fig. 1. Expression of sepF in Anabaena. (A) Schematic of the Anabaena genomic cluster including sepF. (B, C) Detection of sepF expression by northern blot. RNA was extracted from filaments growing in BG11 0 +NH 4 + medium (time 0), transferred to BG11 0 medium and incubated under culture conditions for the indicated times. Hybridization was performed with probes of the sepF gene (B, and upper panel in C), or of rnpB used for normalization (lower panel in C). A quantification of transcript abundance in the ca. 0.6 kb band, normalized for the rnpB signal, is presented in Fig. 3C. Size markers (kb) shown at left of panels are derived from a size standard (RiboRuler High Range, Thermo). (D) Genomic structure of strain CSAV53 (expressing P sepF -mut2-gfp). (E) Spatiotemporal expression from the sepF gene promoter. Filaments of CSAV53 grown in BG11 0 +NH 4 + medium (2 μ g Chl/ml) were transferred to BG11 0 medium and incubated under culture conditions. At the times indicated, filaments were observed by confocal microscopy and photographed. GFP fluorescence (left), cyanobacterial autofluorescence (middle) and brightfield (right) images are shown. Early-differentiating (E), immature (I) or mature (H) heterocysts, the latter exhibiting polar refringent cyanophycin granules, are indicated. Magnification is the same for all micrographs. Images of the WT after 24 h is included as reference. A quantification of fluorescence over 60–88 cells (nondifferentiating cells for times 3–30 h) is presented. A. Valladares et al. Microbiological Research 277 (2023) 127489 6 expressed from the synthetic nitrogen-regulated promoter P ND , which determines basal expression levels in the presence of ammonium, higher levels in the presence of nitrate, and highest levels in the absence of combined nitrogen, in a proportion of ca. 0.1:0.3:1 (Ramos-Le´ on et al., 2015) (Fig. 3A). (It should be pointed out that even segregation of the same P ND -sepF construct substituting for P sepF -sepF in the native locus was not possible.) Additionally, strain CSAV64 bears a gene construct that directs the expression of sepF from the constitutive P trc promoter inserted in the chromosome (see Fig. 4A). Growth of strains CSAV54 and CSAV64 was tested in media with different nitrogen sources. When ammonium-grown filaments of CSAV54 were transferred to medium containing ammonium, nitrate or no combined nitrogen, growth was observed in the three cases. However, whereas in medium with ammonium the exponential growth rate was similar to that of the wild type, growth was impaired in medium with nitrate and, to a larger extent, in the absence of combined nitrogen (Fig. 3B). After prolonged incubation in the latter condition, the cultures of the mutant appeared yellowish, an indication of nitrogen deficiency (Fig. 3C). Regarding the morphology of strain CSAV54, only mild alterations were observed in cultures supplemented with ammonium, where filaments that resembled those of the wild type could frequently be found. In BG11 medium, filament stretches including small cells and others including enlarged cells, generally with aberrant morphology, were observed. In BG11 0 medium, although some filaments with apparently normal heterocysts could be found, many aberrantly enlarged cells, frequently detached from filaments, were also observed (Fig. 3D). When ammonium-grown filaments of strain CSAV64 were transferred to medium containing ammonium, nitrate or no combined nitrogen, growth was similarly impaired in the three media in the fastest growth phase (growth rate ca. half that of the WT) (Fig. 4B). Later, differences with the wild type became smaller in the presence of nitrate or ammonium, but not under diazotrophic conditions (Fig. 4B, C). Regarding the morphology of strain CSAV64, in cultures supplemented with nitrate or ammonium, in addition to apparently normal cells, many aberrant enlarged cells, some of them heavily elongated, were observed Fig. 2. Localization of SepF in Anabaena. (A) Schematic of the genomic structure of strain CSAV52 (sfgfp-sepF). (B) Filaments of strain CSAV52 grown in BG11 0 +NH 4 + (B) or BG11 (C) medium were transferred to BG11 0 medium and incubated under culture conditions. At the time of transfer and after 24 h, filaments were observed under a confocal microscope and photographed. GFP fluorescence (upper panels), cyanobacterial autofluorescence (middle panels) and bright-field (lower panels) images are shown. Arrowheads point to midcell (white) or septal (blue) fluorescence bands. Early-differentiating (E), immature (I) or mature (H) heterocysts, the latter exhibiting polar refringent cyanophycin granules, are indicated. Magnification is the same for all micrographs. A. Valladares et al. Microbiological Research 277 (2023) 127489 7 Fig. 3. Growth and morphology of strain CSAV54 overexpressing sepF from P ND . (A) Schematic of the genetic structure of CSAV54. (B, C) Filaments grown in BG11 0 +NH 4 + medium were used to inoculate, at an initial cell density corresponding to 0.5 μ g Chl/ml, flasks containing BG11 0 , BG11 or BG11 0 +NH 4 + medium, which were incubated under culture conditions. (B) Growth rates were estimated by the increase in OD 750 of cultures. Fast exponential growth corresponded to growth during the first 48 h (see Vel´ azquez-Su´ arez et al., 2020). The values were calculated from data of three independent cultures. The table includes the ratio between the values for CSAV54 and the WT. (C) Flasks were photographed after 4 days of incubation. (D) Filaments from cultures shown in (C) were observed under an optical microscope and photographed. Arrows point to heterocysts. Magnification is the same for all micrographs. A. Valladares et al. Microbiological Research 277 (2023) 127489 8 Fig. 4. Growth and morphology of strain CSAV64 overexpressing sepF from P trc . (A) Schematic of the genomic structure of CSAV64. (B, C) Cells grown in BG11 0 +NH 4 + medium were used to inoculate, at an initial cell density corresponding to 0.5 μ g Chl/ml, flasks containing BG11 0 , BG11 or BG11 0 +NH 4 + medium, which were incubated under culture conditions. (B) Growth rates were estimated by the increase in OD 750 of cultures. Fast exponential growth corresponded to growth during the first 48 h (see Vel´ azquez-Su´ arez et al., 2020). The values were calculated from data of three independent cultures. (C) Flasks were photographed after 7 days of incubation. (D) Filaments from cultures shown in Fig. (C) were observed under an optical microscope and photographed. Arrows point to heterocysts. Magnification is the same for all micrographs. A. Valladares et al. Microbiological Research 277 (2023) 127489 9 (Fig. 4D). In the absence of combined nitrogen, although some normal filaments could be detected, short yellowish filaments and aberrant enlarged cells detached from filaments were also found. Apparently mature heterocysts were also observed, but they were mostly detached from filaments and also larger than in the wild type. In summary, overexpression of sepF in Anabaena is deleterious for growth and produces conspicuous morphological alterations. 3.4. Localization of ZipN under overexpression of sepF ZipN is an essential component of the Anabaena Z-ring, and it is a major tether of FtsZ to the membrane (Camargo et al., 2019). We used the localization of ZipN as a probe of the Z-ring localization in Anabaena. For that, we used a sfgfp-zipN reporter gene expressed from the native zipN promoter (Vel´ azquez-Su´ arez et al., 2022), which was transferred to strain CSAV64, generating strain CSAV73 and, as a control, to the wild type, generating strain CSAV71. Both CSAV71 and CSAV73 also include an intact copy of P zipN -zipN (Fig. 5A). In the wild-type background, ZipN was localized to midcell and the intercellular septa in filaments grown with combined nitrogen, as well as in vegetative cells of diazotrophic filaments, but was absent in heterocysts (Fig. 5B), as reported previously (Valladares et al., 2020). In strain CSAV73, many cells of filaments incubated with ammonium or nitrate presented conspicuous alterations in the pattern of GFP distribution. Alterations included frequently misoriented bands in cells that where themselves misoriented with regard to the filament axis and generally exhibited aberrant shape and increased size (Fig. 5B). Some scattered fluorescence, including peripheral fluorescence, was detected as well (Fig. 5B). In the absence of combined nitrogen, similar alterations were observed in the GFP distribution in vegetative cells of CSAV73, including the presence of short deformed filaments frequently showing a terminal heterocyst. In addition, whereas in the wild-type background GFP fluorescence was detected neither in immature (identified by autofluorescence loss) nor in mature (with polar refringent granules) heterocysts, as previously Fig. 5. Localization of ZipN in filaments overexpressing sepF. (A) Schematic of the genomic structure of strains CSAV71 (WT background) and CSAV73 (P trc -sepF background) in the zipN locus. (B) Cells grown in BG11 medium were used to inoculate, at an initial cell density corresponding to 0.4 μ g Chl/ml, flasks containing BG11 0 , BG11 or BG11 0 +NH 4 + medium, which were incubated under culture conditions. After 25 h, aliquots of cultures were observed by confocal microscopy and photographed. GFP fluorescence (left panels), merged GFP and cyanobacterial autofluorescence (middle panels) and bright-field (right panels) images are shown. Arrowheads point to aberrant GFP signals (see the text). Early-differentiating (E) or mature (H) heterocysts, the latter exhibiting polar refringent cyanophycin granules, are indicated. Magnification is the same for all micrographs. A. Valladares et al.