SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120

ABSTRACT Anabaena sp. PCC 7120 grows by forming filaments of communicating cells and is considered a paradigm of bacterial multicellularity. Molecular exchanges between contiguous cells in the filament take place through multiprotein channels that traverse the septal peptidoglycan through nanopores connecting their cytoplasms. Besides, the septal-junction complexes contribute to strengthen the filament. In search for proteins with coiled-coil domains that could provide for cytoskeletal functions in Anabaena, we identified SepT (All2460). SepT is characteristic of the phylogenetic clade of filamentous cyanobacteria with the ability to undergo cell differentiation. SepT-GFP fusions indicate that the protein is located at the cell periphery and, conspicuously, in the intercellular septa. During cell division, the protein is found at midcell resembling the position of the divisome. The bacterial adenylate cyclase two-hybrid analysis shows SepT interactions with itself and putative elongasome (MreB, RodA), divisome (FtsW, SepF, ZipN), and septal-junction (SepJ)-related proteins. Thus, SepT appears to rely on the divisome for localization at mature intercellular septa to form part of intercellular protein complexes. Two independently obtained mutants lacking SepT showed alterations in cell size and impaired septal and peripheral peptidoglycan incorporation during cell growth and division. Notably, both mutants showed conspicuous alterations in the array of nanopores present in the intercellular peptidoglycan disks, including aberrant nanopore morphology, number, and distribution. SepT appears, therefore, to be involved in the control of peptidoglycan growth and the formation of cell-cell communication structures that are at the basis of the multicellular character of this group of cyanobacteria. IMPORTANCE Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

as in the intercellular mature septa, invoking a role in the formation of septal structures for intercellular communication (32,33).
In most bacteria, the tubulin homolog FtsZ is essential for viability.FtsZ polymerizes into short filaments to form, together with other proteins, a ring at the future site of cell division, the so-called Z-ring.The Z-ring organizes the multiprotein complex termed the divisome, which includes the enzymatic machinery for PG growth to synthesize the new poles of the resulting daughter cells (34).In addition to FtsZ (35,36), cyanobacteria possess the protein ZipN, which represents a central player in divisome assembly (37,38).In Anabaena, ZipN is essential, and it has been identified as a principal tether of FtsZ to the cytoplasmic membrane and divisome organizer (39,40).Anabaena also includes homologs for the divisome components FtsE, FtsK, FtsQ, FtsX, FtsI, FtsW, and SepF [see references (40)(41)(42)].
In Anabaena, the septal-junction-related proteins, SepJ, which itself contains an N-terminal coiled-coil domain and forms multimers (43,44), FraC (18), and SepI (20) are recruited to the intercellular septa through interactions with the divisome during cell division, thus providing a link between cell division and cell-cell communication.In addition, other cyanobacterial CCRPs were identified more recently to perform cytoskeletal and cytoskeletal-like functions in a diverse set of cyanobacterial species (45), including the heteropolymer-forming ZicK and ZacK (46).
In this work, we aimed at the characterization of another coiled-coil-rich protein and deciphering its function in Anabaena as a model of bacterial multicellularity.We found that All2460, which we have termed SepT, is a new element connecting cell division and PG growth to the formation of the septal structures involved in cell-cell adhesion and intercellular communication through the filament.

All2460 predicted domains and phylogenetic distribution
Initially selected in a screen, using COILS (47), searching for coiled-coil-rich proteins with a putative function in the Anabaena multicellularity and morphology (45), All2460 is characterized by three distinct coiled-coil-rich regions as well as two N-terminal transmembrane domains (TMDs, residues 12-50, predicted with TMHMM) (Fig. 1A and B).Using the Conserved Domain Search (CD Search, NCBI), All2460 is predicted to contain a structural maintenance of chromosomes (SMC) and a TerB-C domain, suggesting a metal-dependent function in chromosome biology (Fig. 1A).Predictions of the putative localization of the C-terminal non-TMD parts consistently placed the C-terminus in the cytoplasm (predicted using PSIPRED, PSORTb, and Gneg-mPLoc).Using AlphaFold (48), All2460 is predicted to form a homodimer (Fig. 1C).A further search for All2460 homologs using amino acid sequence similarity shows that all tested filamentous heterocyst-forming cyanobacteria encode a protein homologous to All2460 (Data Set S1; Fig. S1; Fig. 1D).Outside the heterocyst formers, there are some homologs that using our thresholds are clustered in the same protein family as All2460.These homo logs are found in filamentous non-heterocystous cyanobacteria such as Spirulina, and closely related genera to the order Nostocales such as Gloeocapsa, Chroococcidiopsis, or Pleurocapsa (49).Moreover, the protein sequence alignment of SepT homologs (Fig. S1) indicates a high-sequence divergence between homologs from the heterocyst-forming and non-heterocystous cyanobacteria, suggesting also functional divergence.
To further study the genomic neighborhood of All2460, we searched for the presence of conserved gene order at the All2460 locus using CSBFinder-S (50).Our results reveal a conserved synteny block that includes two genes next to all2460, namely all2459 and all2461 (Fig. 1E).Unlike All2460, both All2459 and All2461 are not predicted to contain TMDs but both are predicted to contain P-loop NTPase domains, which are commonly found in MinD, ParA, and other DNA partitioning systems (51).All three genes are about twofold upregulated 21 hours after nitrogen stepdown (52), suggesting some involve ment in diazotrophic growth.

All2460 localization in Anabaena
We studied the localization of All2460 in Anabaena by means of fusions to GFP.For that, a replicative plasmid encoding All2460 C-terminally fused to GFP, directed by the native promoter sequence, was transferred to Anabaena wild type (WT), generating strain CSCV25, and to strain BS1 (Δall2460) (see below), generating strain CSCV26.In both strains, CSCV25 and CSCV26, GFP fluorescence was detected in the cell periphery and, conspicuously, in the intercellular septa between contiguous cells along the filament (Fig. 2).Remarkably, midcell GFP bands were detected in some dividing cells, matching the localization of the progressing new septum.When the N-terminal transmembrane domain of SepT was deleted, the resulting protein fused to GFP produced fluorescence in patches without any specific localization in the cell (Fig. S2), suggesting that mem brane anchorage is essential for the correct localization.These observations suggest that All2460 is a new septal protein of Anabaena, and accordingly, we term it SepT.
To identify further interaction partners of SepT, we performed co-immunoprecipita tions of Anabaena WT expressing SepT-GFP and analyzed co-precipitated proteins by LC-MS/MS.With this approach, we were able to verify the interaction of SepT with MreB and SepJ.Additionally, proteins that co-precipitated with SepT included All4981 and All2459, from its own synteny block, and three different penicillin-binding proteins (PBPs): All2981, All2952, and Alr0718 (the full list of interactions identified is supplied in Data Set S2).

Inactivation of sepT
To study the function of SepT, we sought the generation of Anabaena derivatives lacking a functional all2460 ORF.Strain CSCV9, generated in the Seville lab, bears gene-cassette C.K1, encoding resistance to kanamycin and neomycin, inserted into all2460 (Fig. 4A).Growth of this mutant was studied in media supplemented with nitrate or lacking combined nitrogen (Fig. 4B).It was able to grow under the two conditions, exhibiting growth rates similar to those of Anabaena WT both in the exponential growth phase and in the phase of slow growth (Fig. 4B).Consistent with these results, strain BS1, which was previously generated in the Kiel lab, bearing gene cassette C.S3 substituting for all2460 (Fig. 4A), also exhibited growth rates comparable to those of Anabaena WT (Fig. 4B).In the absence of combined nitrogen, both CSCV9 and BS1 exhibited mature heterocysts with conspicuous polar granules, as is the case for Anabaena WT (Fig. 5A).
The cell area and, as an estimation of the cell morphology, the aspect ratio (the ratio between the cell axis parallel to the filament and the axis perpendicular to the filament) were determined in strains CSCV9 and BS1.Because strains maintained in different labs have frequently been noticed to exhibit some different phenotypic characteristics, each of the two mutants was compared to the respective Anabaena WT strain from which they were derived (WT-s, WT from Seville; WT-k, WT from Kiel).While actively growing cells (48 hours) of CSCV9 were smaller than those of its parental strain in the presence and absence of combined nitrogen (Fig. 5A and B) (Student's t-tests indicate significant differences, P < 0.01, for comparison of WT-s and CSCV9 with NO 3 -or N 2 after 48 hours of incubation; and nonsignificant differences, P > 0.05, after 216 hours), cells of BS1 appeared larger than those of its parental strain (Fig. 5A and B).(Student's t-tests indicate significant differences, P < 0.01, for comparison of WT-k and BS1 with NO 3 -or N 2 after 48 or 216 hours of incubation.)The alterations in cell size in CSCV9 and BS1 suggest that both strains suffer a certain discoordination between cell growth and division.In contrast to C.K1 introduced in CSCV9, the C.S3 gene cassette introduced in BS1 bears strong transcriptional terminators, which could affect the expression of the downstream ORF all2459.This could contribute to explain the differences in the effects of the mutations in CSCV9 and BS1.
Finally, cell aspect ratio determinations indicated that the morphology of strains CSCV9 and BS1 is similar to that of their respective parentals (Fig. 5C).(Student's t-tests indicate significant differences, P = 0.002, only for comparison of CSCV9 and its WT after 216 hours of incubation with NO 3 -.)

Localization of PG growth in sepT mutants
The interaction of SepT with some proteins involved in lateral or septal PG growth, as suggested by the BACTH assays, led us to check whether the pattern of PG growth was affected by the mutation of sepT.For that, we used labeling with fluorescent vancomycin (Van-FL), which highlights the sites of PG incorporation, as done before in Anabaena [ (22); see also reference (32)].Filaments of Anabaena WT and strains CSCV9 and BS1 were incubated in BG11 or BG11 0 for 48 (exponential growth) and 216 hours (slow growth) under the same conditions used for the determination of growth rates and labeled with Van-FL.In Anabaena WT, cells from BG11 cultures, as well as vegetative cells from BG11 0 medium, presented weak peripheral and strong septal labeling with alternating intensi ties (lower in the more recently formed septa than the older ones), as well as labeling in the septum under construction during cell division (Fig. 6), as previously described [see references (32,33)].Filaments of CSCV9 and BS1 incubated in BG11 or BG11 0 medium showed a labeling pattern similar to that of the WT (Fig. 6A and B).However, quantifica tion of Van-FL fluorescence intensity in the cell periphery (lateral fluorescence) and in the septal regions indicates that after 48 hours in BG11 medium, both lateral and septal fluorescence were lower in the mutants than in the WT (Student's t-tests P < 0.01), becoming more similar after 216 hours [at 216 hours only lateral fluorescence in BS1 appeared significantly lower (P < 0.01) than in the WT] (see the Table in Fig. 6A).In BG11 0 medium, fluorescence intensity in vegetative cells appeared similar in the mutants and the WT [only lateral fluorescence at 216 hours was significantly lower (P < 0.01) in CSCV9 than in the WT] (see the Table in Fig. 6B).

Localization of MreB, MreC, and MreD in strain CSCV9
We also studied whether inactivation of sepT affected the localization of the elongasome components MreB, MreC, and MreD.For that, we transferred gene constructs, whereby the mreBCD promoter region directs the expression of the fusion proteins sfGFP-MreB, sfGFP-MreC, or sfGFP-MreD to strain CSCV9, generating strains CSCV11, CSCV12, and CSCV13, respectively.Strains CSCV6, CSCV7, and CSCV8 express the same fusion proteins, respectively, in the WT background (32).The six reporter strains include, in addition, the intact mreBCD operon in its native genomic locus.In the presence of combined nitrogen, strains CSCV11, CSCV12, and CSCV13 exhibited GFP fluorescence localized through the cell periphery, including the intercellular septal regions, and at midcell, matching the divisome localization in dividing cells (Fig. 7A), a distribution similar to that described for strains, CSCV6, CSCV7, and CSCV8, respectively (32; Fig. 7A).Upon N-stepdown, vegetative cells of CSCV11, CSCV12, and CSCV13 exhibited peripheric, septal, and midcell GFP fluorescence similar to the pattern found in filaments incubated with nitrate.Heterocysts showed peripheric fluorescence and, frequently, fluorescence spots focalized at the cell poles (Fig. 7B).These observations also match the distribution observed in CSCV6, CSCV7, and CSCV8, respectively (Fig. 7B; 33), indicating that localization of MreB, MreC, and MreD is not noticeably affected in the sepT mutant background.

Morphology of the septal nanopore structures in sepT mutants
Given that alterations in the number and morphology of septal nanopores have been described in mutants of other septal proteins in Anabaena (e.g., 13), we isolated PG sacculi from the sepT mutant strains CSCV9 and BS1 and observed the septal disks by TEM (Fig. 8; Fig. S3).In comparison to the WT, we observed a heterogeneity in the array of septal disk nanopores in the mutants.Septal disks in the WT contain an average of ca.40 nanopores of about 17 nm in diameter concentrated in the central part of the disk (13; see Fig. 8).In comparison to the WT, septal disks of both CSCV9 and BS1 generally contained fewer nanopores with a slightly smaller size (Fig. 8B).However, although in some cases the mutant disks showed a nanopore distribution that resembles the WT pattern (first line in Fig. 8A), disks with nanopores distributed throughout the whole disk area (second line in Fig. 8A) or including only two to four, larger than average, nanopores (third line in Fig. 8A) were also observed.In addition, some disks showing pores severely enlarged and of abnormal (non-circular) shape, apparently resulting from PG mesh breakage, were also frequently detected in both mutants (fourth line in Fig. 8A). Figure S3 shows that peptidoglycan isolated from filaments of BS1 grown on agar plates presented a distribution of septa similar to that found in liquid medium.

DISCUSSION
In this work, we have identified the product of Anabaena ORF all2460, a cyanobacterial cytoskeletal protein linked to the divisome, the elongasome and the septal-junctions, which we have termed SepT.SepT is predicted to be a coiled-coil-rich protein characteris tic of filamentous cyanobacteria capable of heterocyst differentiation.Indeed, homologs to SepT have been found encoded in the genomes of all tested heterocyst-forming strains.As in most cases, Anabaena SepT is predicted to be an inner membrane-anchored cytoplasmic protein and as most CCRPs, it appears able of self-interactions (Fig. 3B), with predicted formation of structured dimers (Fig. 1C).
In Anabaena, SepT is localized in the cell periphery and the intercellular septa, and in dividing cells, it is also detected at midcell matching the localization of the divisome (Fig. 2).Localization at the cell periphery and the divisome has also been described for the elongasome components MreB, MreC, and MreD in Anabaena (32).Moreover, we have detected SepT interactions with MreB (Fig. 3B), invoking a role of SepT in the elongasome function.We have not detected any apparent alteration of MreB, MreC, or MreD localization in the absence of a functional SepT protein (Fig. 7).However, two different sepT mutants showed impaired regulation of cell size (Fig. 5B), which might reflect a discoordination between cell growth and division, and altered PG growth with lower incorporation, especially under conditions of higher growth rate (exponential growth with combined nitrogen) in the mutants (Fig. 6A).In addition, we have detected interactions of SepT with putative RodA (Fig. 3A), the elongasome glycosyl-transferase catalyzing PG polymerization, as well as with three putative PBPs (All2981, All2952, and Alr0718).These results suggest a role for SepT in the regulation of PG expansion during cell growth.n, sample size (number of septal disks or nanopores, respectively).The difference between each mutant and the wild type was assessed by the Student's t-test (P values are indicated).For comparisons between the two mutant strains, P values were 0.4933 for nanopore number and 0.5872 for nanopore diameter.
Data on Anabaena WT are from reference (13).
The localization of SepT in the intercellular septa and the divisome coincides with that reported for other septal proteins that play important roles in filament maintenance and intercellular communication in Anabaena, including SepJ, FraC, and SepI.Thus, SepT could have a function related to the specialized structure of the intercellular septa of heterocyst-forming cyanobacteria.Notably, our past and current investigations suggest that interaction with the divisome during cell division is a mechanism for the localization of proteins that remain in the intercellular septa once the division event has concluded.Indeed, SepJ has been shown to interact with the divisome components FtsQ and ZipN (40,43), and also with SepI, which interacts with FtsI, SepF, and ZipN (20).Here, we have detected clear interactions of SepT with ZipN, SepF, and FtsW.This, together with its localization to the divisome, points to a similar mechanism for SepT localization to the intercellular septa.
The interaction of SepT with putative FtsW (divisome PG glycosyl-transferase) suggests an effect on septal PG growth or remodeling.Indeed, we have observed conspicuous alteration on the nanopore array of the septal PG disks in two different sepT mutants, including a lower average nanopore number, altered nanopore distribution, and even very aberrant septal disks with only two to four large nanopores or showing apparent tears in the PG mesh (Fig. 8).SepJ, with which SepT also interacts (Fig. 3B), is also a coiled-coil-containing protein that influences the septal disk nanopore array in Anabaena, although sepJ inactivation had milder effects than sepT inactivation leading to reduced nanopore numbers (15).In conclusion, besides influencing lateral PG growth, SepT is a new component of the specialized septal structure of filamentous heterocystforming cyanobacteria directly linked to the cell growth and cell division machineries.It has a role in the processing of the intercellular PG required for the formation of intercellular communication structures that are key feature of multicellular organisms.

Bacterial strains and growth conditions
Anabaena sp.PCC 7120 and mutant strains were grown photoautotrophically in BG11 medium (containing NaNO 3 ) or in BG11 0 (lacking combined nitrogen) (6), or in BG11 0 supplemented with 4 mM NH 4 Cl and 8 mM TES-NaOH buffer, pH 7.5 at constant illumination of 12-30 photons m −2 s −1 intensity.Cells were either grown in Erlenmeyer flasks with shaking or on medium solidified with 1% (wt/vol) Difco agar.For the mutants, media were supplemented with antibiotics: spectinomycin (Sp) and streptomycin (Sm) at 5 µg mL −1 each in solid media or at 2 µg mL −1 each in liquid media, or with neomycin (Nm) at 25 µg mL −1 in solid media or 5 µg mL −1 in liquid media.Chlorophyll content of the cultures was determined after extraction with methanol (53).In Anabaena, 1 µg chlorophyll corresponds to ca. 3.3 × 10 6 cells (54).(Table S1 and S2 list all used cyanobac terial strains, plasmids, and oligonucleotides.)

Distribution of homologs in cyanobacteria
Homologs to SepT were detected in completely sequenced genomes publicly available in RefSeq (version 01/2021) by amino acid sequence similarity using stand-alone BLAST (55, 56) (v.2.2.26).Protein sequences that were found as BLAST hits with a threshold of E-value ≤1×10 −5 were further compared to SepT by global alignment using needle (57).Hits having ≥30% identical amino acids in the global alignment were considered as homologs.For the phylogenetic analysis, homologs in a set of representative heterocys tous species were manually selected and complemented by all homologs in non-heter ocystous species.Amino acid sequences were aligned with MAFFT (v.7.475) (58).The phylogenetic tree was inferred with IQ-TREE (59) (v.1.6.12)with restricted automatic model selection to the Le & Gascuel model ( 60) and fast bootstrap with 1,000 replicates.The tree was rooted using the MAD approach (61).The phylogenetic tree was visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

Plasmid and Anabaena mutant construction
Mutant strain CSCV9 carries a version of the sepT gene in which codons 69-367 have been substituted by gene-cassette C.K1 encoding Km/Nm resistance.To generate it, two DNA fragments were amplified from Anabaena genomic DNA using the primer pairs all2459-1/all2460-2 (encompassing sequences internal and upstream of sepT) and all2460-1/all2461-1 (encompassing sequences internal and downstream of sepT), including terminal restriction sites BamHI and PstI.Both fragments were joined together by overlapping PCR, and the resulting single fragment was cloned into mobilizable vector pRL277 [encoding the gene sacB for positive selection (69)].Gene-cassette C.K1 from plasmid pRL161 (70) was then inserted into the internal BamHI site, generating plasmid pCSCV36, which was transferred to Anabaena by conjugation (71).
Mutant BS1 carries gene cassette C.S3, encoding Sm/Sp resistance, substituting for the sepT gene.To generate it, 1,500 bp upstream and downstream of the sepT ORF were amplified by PCR from Anabaena genomic DNA using primers 842KO_2A/842KO_2B and 842KO_4A/842KO_4B, respectively.The upstream and downstream sepT regions flanking the C.S3 cassette [amplified with primers CS.3_Fwd/CS.3_Revusing pCSEL24 (72) as a template] were then inserted into PCRamplified pRL278, including the gene sacB for positive selection (73), using primers pRL271_Fwd/pRL271_Rev by Gibson assembly, yielding pTHS109, which was transferred to Anabaena by conjugation.For both CSCV9 and BS1 mutants, insertion of the mutagenic construct by double crossover was selected by resistance to sucrose and lack of antibiotic resistance encoded in the vector portion of the transferred plasmid.Both mutants were segregated for the mutant chromosomes, as tested by PCR analysis (not shown).
For the generation of strains CSCV11, CSCV12, and CSCV13, strains expressing a sfgfp-mreB, sfgfp-mreC, or sfgfp-mreD fusion gene, respectively, expressed from the native mreBCD operon promoter (P mreB ), strains CSCV6, CSCV7, and CSCV8, which, respectively, express the same fusion genes in the WT background, were used as recipient of plasmid pCSCV36.Insertion of the mutagenic construct and segregation for the mutant chromosomes were verified by PCR analysis as above.
The replicative plasmid pTHS240, used for the localization of SepT-GFP, included a sepT-gfpmut3.1 fusion gene under the control of the native promoter (predicted by BPROM).To generate it, the sepT promoter sequence and ORF were amplified using the primer pair p842_25C_long_A/Nos842_2B; the gfpmut3.1 sequence was amplified using the primer pair GFP_842_A/GFP_25C_R, and both PCR products were cloned into PCRamplified plasmid pRL25C (74) using primer pair pRL25C_F/pRL25C_R by Gibson assembly.
The replicative plasmid pTHS143, driving the expression of SepT-GFP from P petE used for co-IP analysis, was cloned as follows: the petE promoter was amplified using primer pair petE_903_Fwd/ pRL25c_NEB_Rev; sepT was amplified using primer pair Nos842_2 A/ Nos842_2B, and gfpmut3.1 was amplified using pRL25c_NEB_Fwd/ pRL25c_NEB_Rev, and all three PCR products were cloned into PCRamplified pRL25C using primer pair pRL25c-903_V_F/ pRL25c-903_V_R.
β-galactosidase activity was measured after growth in liquid medium in the presence of IPTG and antibiotics, using ο-nitrophenol-β-galactoside as a substrate.Either the ο-nitrophenol produced per milligram of protein versus time was represented and the β-galactosidase activity deduced from the slope of the linear function, or the ο-nitrophe nol produced was recorded in Miller units as described in the manufacturer's manual (Euromedex).

Mass spectrometry analysis
Mass spectrometry was performed as previously described (20).The acquired MS/MS data were searched with the SequestHT algorithm against the entire reviewed Uniprot protein database of Anabaena sp.PCC 7120, including proteins encoded in plasmids (6,922 sequences in total).Static modification applied was carbamidomethylation on cysteine residues, while oxidation on methionine residues was set as dynamic modifica tion.Spectra were searched with full-enzyme specificity.An MS mass tolerance of 10 ppm and an MS/MS tolerance of 0.02 Da were used.Proteins were identified with at least three unique peptides with a FDR confidence ≤0.01 (high).

Van-FL staining and quantification
Filaments were stained by incubation with 2 µg mL −1 Vancomycin-FL (Bodipy-FL conjugate, Invitrogen), and lateral and septal fluorescence were quantified with ImageJ (76) processing of fluorescence images by collecting total fluorescence in manually defined equal square sections, as described (32).For each cell, lateral fluorescence was calculated as the mean of the values of four sections and septal fluorescence as the mean of two sections, one at each cell pole.Twenty to thirty cells were counted for each strain and condition, and the average values were calculated.

PG sacculi isolation and visualization
PG was isolated from filaments grown in BG11 medium by protease treatment and hot detergent extraction, as described (15).Aliquots of the obtained preparations were deposited on formvar-carbon filmcoated copper grids and stained with 1% (wt/vol) uranyl acetate and examined with a Zeiss Libra 120 plus (Zeiss) transmission electron microscope at 120 kV.

ADDITIONAL FILES
The following material is available online.

FIG 1
FIG 1 SepT domain distribution and phylogeny.(A) Prediction of conserved domains in SepT using the Conserved Domain Search (NCBI).Orange bars indicate the presence of the two transmembrane domains; a structural maintenance of chromosomes domain is depicted in gray, and a TerB-C domain (putative metal chelating) domain is shown in yellow.The SMC and TerB-C domains overlap in part, which is highlighted by a mixed gray-yellow part.(B) Prediction of coiled-coil-rich regions using the COILS algorithm and three different screening windows for repetitive heptamer sequences (windows 14, 21, and 28).(C)Prediction of the structure of a SepT dimer (colored by pLDDT) according to AlphaFold algorithm.(D) Phylogenetic tree of selected SepT homologs.Heterocyst-forming species are written in black, and non-heterocystous species are written in blue.Nostoc sp.PCC 7120 is the same strain as Anabaena sp.PCC 7120.Bootstrap values are shown next to ancestral node, and the branch width is scaled to the bootstrap values.(E) The genomic region of all2460 taken from the Integrated Microbial Genomes and Microbiomes of the Joint Genome Institute database.

FIG 3
FIG 3 Analysis of SepT interactions by BACTH.The topology of each fusion is indicated by the order of components (T18-protein and T25-protein denote the corresponding adenylate cyclase domain fused to the N-terminus of the tested protein, whereas protein-T18 and protein-T25 denote fusions to the C-terminus).(A) Interaction of protein pairs produced in Escherichia coli was assayed by measurements of β-galactosidase activity (nmol ONP min −1 mg protein −1 ) in liquid cultures incubated at 30°C.Data are the mean and standard deviation of two to nine determinations of the activity with the indicated protein fused to T25 (or the empty vectors pKNT25 or pKT25) and SepT-T18 (dark bars), or the indicated protein (or the empty vectors pKNT25 or pKT25) and pUT18C (clear bars); or with the indicated protein fused to T18 (or the empty vectors pUT18C or pUT18) and SepT-T25 (dark bars), or the indicated protein (or the empty vectors pUT18C or pUT18) and pKT25 (light bars).Significance of differences was assessed by Student's t-tests.Asterisks indicate strains expressing a pair of tested proteins that exhibited β-galactosidase activity significantly different (P < 0.023) from the two controls: the strains expressing each fused protein and containing the complementary empty vector.(B) E. coli cells were subjected to β-galactosidase assay in triplicates from three independent colonies after grown in liquid cultures at 20°C for 2 days.Quantitative values are given in Miller units, and the mean results from three independent colonies are presented.Negative: N-terminal T25 fusion construct of the respective protein co-transformed with empty pUT18C.Positive: Zip/Zip control.Error bars indicate standard deviations (n = 3).Values indicated with asterisks are significantly different from the negative control.***P < 0.001, ****P < 0.0001 (Dunnett's multiple comparison test and one-way ANOVA).

FIG 4
FIG 4 Genomic structure and growth of strains with inactivated sepT.(A) Schematic of the genetic structure of strains CSCV9 and BS1 in the sepT genomic region.(B) Filaments of strains Anabaena WT, CSCV9, and BS1 were grown in BG11 medium, transferred to BG11 (containing NaNO 3 − ) or BG11 0 (no combined nitrogen) at a cell density corresponding to 0.5 μg Chl mL −1 , and incubated under culture conditions.At the indicated times, the OD 750 (At) was measured in aliquots of each culture.The values of two independent cultures of each condition (one culture of CSCV9 under N 2 ) were represented and adjusted to sequential linear functions.A0 represents the OD 750 at the start of the culture.Growth rate constant, μ (day −1 ), corresponds to ln2/t d , where t d is the doubling time, calculated from the increase in OD 750 from 0 to 98.5 hours (exponential growth) and from 170.5 to 266.5 hours (slow growth) of incubation as above.Mann-Whitney tests indicated no significance of differences (P > 0.1) between each mutant and the WT for any time and condition.

FIG 5
FIG 5 Cell size and morphology of sepT mutants.Filaments of Anabaena WT (WT-s, the parental for CSCV9; WT-k, the parental for BS1), CSCV9 (sepT::C.K1), and BS1 (sepT::C.S3) grown in BG11 medium were transferred to BG11 or BG11 0 , at a cell density corresponding to 0.5 µg Chl mL −1 , and incubated under culture conditions.(A) After 48 hours, filaments from BG11 0 cultures were photographed.Purple arrows point to polar granules in heterocysts.Magnification is the same for all micrographs.(B and C) After 48 and 216 hours, aliquots of each culture were photographed and used for cell area (B) and aspect ratio (C) determinations.Two hundred to three hundred cells (vegetative cells in the diazotrophic cultures) from two different cultures of each strain and condition (150 cells for WT-k) were measured.The aspect ratio is the result of dividing the length of the axis parallel to the filament by the length of the axis perpendicular to the filament.Notched boxplot representations of the data are shown.The mean values are represented by black dots.Significant differences (P < 0.01), assessed by Student's t-tests, are indicated by **.

FIG 6
FIG 6 Localization of PG growth in sepT mutants.Strains Anabaena WT, CSCV9, and BS1 grown in BG11 medium were transferred (at a cell density of 0.5 µg Chl mL −1 ) to BG11 (A) or BG11 0 (B) medium and incubated under culture conditions.After 48 hours, samples of filaments were stained with Van-FL and observed under a fluorescence microscope and photographed.Van-FL fluorescence (green), cyanobacterial autofluorescence (magenta), and brightfield images are shown.White arrows point to heterocysts.Magnification is the same for all micrographs.After 48 and 216 hours, lateral and septal fluorescence were quantified as described in Materials and Methods.Student's t-test was used to assess significance of differences (Data Set S3).Significant differences (P < 0.01) are indicated by **.

FIG 8
FIG 8 Septal nanopore array in sepT mutant strains.(A) Representative transmission electron microscopy images of PG sacculi septa from Anabaena WT and the sepT mutant strains CSCV9 and BS1 grown in BG11 medium (see Materials and Methods for details).Disks showing abnormal nanopore distribution (second line), few, larger than average, nanopores (third line), or nanopores severely enlarged and of abnormal shape (fourth line) are shown.(B) Number of nanopores per septum and mean nanopore diameter (nm).