The Role of MreB, MreC and MreD in the Morphology of the Diazotrophic Filament of Anabaena sp. PCC 7120

Citation: Velázquez-Suárez, C.; Luque, I.; Herrero, A. The Role of MreB, MreC and MreD in the Morphology of the Diazotrophic Filament of Anabaena sp. PCC 7120. Life 2022,12, 1437. https://doi.org/ 10.3390/life12091437 Academic Editors: Asunción Contreras and Jose I. Labella Received: 28 July 2022 Accepted: 9 September 2022 Published: 15 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). life Article The Role of MreB, MreC and MreD in the Morphology of the Diazotrophic Filament of Anabaena sp. PCC 7120 Cristina Velázquez-Suárez, Ignacio Luque and Antonia Herrero * Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, 41092 Seville, Spain *Correspondence: herrer[email protected] Abstract: The cyanobacterium Anabaena sp. PCC 7120 forms filaments of communicating cells. Under conditions of nitrogen scarcity, some cells differentiate into heterocysts, allowing the oxygen-sensitive N 2 -reduction system to be expressed and operated in oxic environments. The key to diazotrophic growth is the exchange of molecules with nutritional and signaling functions between the two types of cells of the filament. During heterocyst differentiation, the peptidoglycan sacculus grows to allow cell enlargement, and the intercellular septa are rebuilt to narrow the contact surface with neighboring cells and to hold specific transport systems, including the septal junction complexes for intercellular molecular transfer, which traverse the periplasm between heterocysts and neighboring vegetative cells through peptidoglycan nanopores. Here we have followed the spatiotemporal pattern of peptidoglycan incorporation during heterocyst differentiation by Van-FL labeling and the localization and role of proteins MreB, MreC and MreD. We observed strong transitory incorporation of peptidoglycan in the periphery and septa of proheterocysts and a maintained focal activity in the center of mature septa. During differentiation, MreB, MreC and MreD localized throughout the cell periphery and at the cell poles. In mreB,mreC or mreD mutants, instances of strongly increased peripheral and septal peptidoglycan incorporation were detected, as were also heterocysts with aberrant polar morphology, even producing filament breakage, frequently lacking the septal protein SepJ. These results suggest a role of Mre proteins in the regulation of peptidoglycan growth and the formation of the heterocyst neck during differentiation, as well as in the maintenance of polar structures for intercellular communication in the mature heterocyst. Finally, as previously observed in filaments growing with combined nitrogen, in the vegetative cells of diazotrophic filaments, the lack of MreB, MreC or MreD led to altered localization of septal peptidoglycan-growth bands reproducing an altered localization of FtsZ and ZipN rings during cell division. Keywords: heterocyst differentiation; heterocyst neck; intercellular communication; Mre proteins; peripheral peptidoglycan growth; polar peptidoglycan growth 1. Introduction Filamentous heterocyst-forming cyanobacteria, such as the model strain Anabaena sp. PCC 7120 (hereafter Anabaena) represents genuine pluricellular bacteria in which the unit for selection has shifted from the single cell to the filament [ 1 , 2 ]. The Anabaena filament is composed of hundreds of cells, each of which is surrounded by an individual cytoplasmic membrane, whereas the outer membrane is continuous along the filament, defining a commonly shared periplasm. Indeed, the continuous periplasm has been considered a path that allows intercellular molecular exchange through the filament [ 3 ]. In addition, neighboring cells are communicated through septal junction complexes composed of polytopic cytoplasmic-membrane anchored proteins [ 4 , 5 ] that traverse the septal peptidoglycan (PG) by perforations termed nanopores, which are drilled by PG amidases [6–8]. Regarding the PG sacculus, the continuous filament periplasm contains a single molecule that completely encircles each cell and is engrossed in the septal location between contiguous cells [ 9 ]. In relation to its multicellular structure, Anabaena presents distinct Life 2022,12, 1437. https://doi.org/10.3390/life12091437 https://www.mdpi.com/journal/life Life 2022,12, 1437 2 of 15 characteristics in the pattern of PG incorporation. During cell division, septal incorporation occurs in the divisome to build the new septa of the resulting daughter cells. In addition, weak peripheral PG growth elongates the cells during cell growth, and continuous PG incorporation occurs in the intercellular locations along the filament, which persists after the division has been completed [ 10 – 12 ]. In model unicellular rod-shaped bacteria, the proteins MreB, MreC and MreD are involved in the spatial localization and regulation of the enzymatic machinery for lateral PG growth organized in the multiprotein complex termed the elongasome. MreB is an actin structural homolog that forms dynamic polymers associated with the inner face of the cytoplasmic membrane. MreC is a cytoplasmic membrane-anchored protein with most of its extension residing in the periplasm, and MreD is an integral membrane protein [ 13 ]. Notably, in Anabaena, MreB, MreC and MreD have been localized all around the cell periphery, including the mature intercellular septa, and in the divisome through all stages of cell division, having been implicated in the regulation of peripheral, medial (divisome-associated) and septal PG incorporation [10]. Depending on environmental conditions, the Anabaena filament can include different cell types specialized in specific functions. Thus, heterocysts are cells specialized in the fixation of atmospheric nitrogen that differentiates at semi-regular intervals along the filament under nitrogen deprivation conditions. Heterocysts supply vegetative cells with organic nitrogen. Vegetative cells fix CO 2 through oxygenic photosynthesis and provide the heterocysts with organic carbon to be utilized as a source of reductants and as carbon skeletons for the incorporation of the ammonium resulting from N2fixation [14,15]. Because the enzymatic machinery for N 2 reduction to ammonium is extremely O 2 sensitive, a main issue in heterocyst performance is the maintenance of a microoxic internal milieu, to which the presence of extra glycolipid and polysaccharide envelope layers, deposited during the differentiation process, contribute (see [ 16 ]). However, the entrance of N 2 into the heterocysts is needed. Key to regulating gas exchanges is the differentiation of the heterocyst cell poles to form the specialized structure known as the heterocyst neck, which narrows the contact space between the heterocyst and the adjacent vegetative cells and represents the main path for gas exchange [ 14 , 17 ]. Indeed, intercellular molecular exchange involving heterocysts is thought to take place to a good extent through the heterocyst neck. Thus, although some membrane transporters of different protein families are localized through the heterocyst periphery, some others, such as the ABC-type exporters HetC, possibly involved in the export of morphogens from the heterocyst [ 18 , 19 ], and DevBCD-HgdD, for heterocyst glycolipid export [ 20 ], preferentially localize at the heterocyst poles. Additionally, the pentapeptide-repeat protein HglK [ 21 ] and the septal-junction associated proteins SepJ [ 22 ] and FraCD [ 23 ] are localized to the heterocyst-vegetative cell septa. Finally, the ABC-permease protein FraE [ 24 ] and the TonB-like protein SjdR [ 25 ] have been identified as required for the constriction and arrangement of the heterocyst polar structures. Besides peripheral PG growth required for cell enlargement, the differentiation of the heterocyst neck also appears to involve PG remodeling/turnover see [ 11 ]. Consistent with this, mutations in genes encoding enzymes involved in different steps of PG synthesis lead to impaired heterocyst differentiation [ 26 – 29 ]. Moreover, PG septal disk diameter, nanopore diameter and nanopore number are larger in septa between vegetative cells and heterocysts than in septa between two vegetative cells [ 24 ], and mutants deficient in the PG amidases AmiC1 [7] or AmiC3 [8] are unable of diazotrophic growth. In this work, we have monitored the incorporation of PG and the localization of the MreB, MreC and MreD proteins in Anabaena during heterocyst differentiation and in mature diazotrophic filaments. We have also addressed the influence of MreB, MreC and MreD on peripheral, medial and septal PG growth, the filament geometry and the formation of heterocyst-vegetative cell connections. Life 2022,12, 1437 3 of 15 2. Materials and Methods 2.1. Strains and Growth Conditions Anabaena sp. strain PCC 7120 and mutant strains (strains and plasmids used in this work are listed in Table S1) were grown in BG11 medium (containing NaNO 3 as a nitrogen source) [ 30 ] containing ferric citrate instead of ferric-ammonium citrate. Cultures were incubated at 30 ◦ C with illumination (12 µ E m −2 s −1 white light from Osram LED lamps 16.4 W/4000 K), in Erlenmeyer flasks with shaking (100 rpm) or in plates of medium solidified with 1% Difco agar. For the mutants, media were supplemented with antibiotics: spectinomycin dihydrochloride pentahydrate (Sp) and streptomycin sulfate (Sm) at 5µg mL−1 each in solid medium or 2.5 µ g mL −1 each in liquid medium (CSCV2, CSCV6, CSCV7, CSCV8, CSSC19, CSAV39), with neomycin sulfate (Nm) at 25 µ g mL −1 in solid medium or 5 µ g mL −1 in liquid medium (CSS89, CSCV1, CSCV4), or with Sm, Sp and Nm (CSCV14, CSCV15, CSCV16, CSCV17, CSCV18, CSCV19, CSCV20, CSCV21, CSCV22). For the experiments described in this work, BG11-grown filaments of the indicated strains were transferred to (at a cell density corresponding to 0.5 µ g chlorophyll mL −1 ) and incubated in BG11 0 medium [ 30 ] lacking NaNO 3 and antibiotics. Chlorophyll a(Chl) content of the cultures was determined after extraction with methanol [ 31 ]. In Anabaena, 1 µ g Chl corresponds to ca. 3.3 ×106cells [32]. 2.2. Strain Constructions Strains CSCV17, CSCV18 and CSCV19 bear a sepJ-gfpmut2 fusion gene in a mreB,mreC or mreD mutant background, respectively. To generate strain CSCV19, the conjugative plasmid pCSVT22 (encoding Nm resistance and including sequences of the 3’ part of Anabaena sepJ fused to gfpmut2) [ 23 ] was transferred to strain CSCV2 (mreD) by conjugation, and one clone in which the plasmid was inserted by single cross-over into sepJ was selected and verified by PCR. To generate strains CSCV17 and CSCV18, the insert of pCSVT22 was amplified with oligonucleotides alr2338-BamH1 and gfp-BamH1 (listed in Table S1) and cloned in the conjugative vector pCSV3 encoding Sm/Sp resistance [ 33 ] generating plasmid pCSCV38, which was transferred to strains CSCV1 (mreB) and CSCV4 (mreC) by conjugation. Clones in which pCSCV38 was inserted by single cross-over into sepJ were selected and verified by PCR. 2.3. Microscopy For Van-FL labeling and detection, filaments were treated as described [ 10 ] and visualized with a Leica DM6000B fluorescence microscope and a FITCL5 filter (excitation band-pass, 480/40; emission band-pass, 527/30), and photographed with an ORCA-ER camera (Hamamatsu). GFP fluorescence was monitored with an Olympus TCS SP2 confocal laser-scanning microscope equipped with an HCX PLAN-APO 63 × 1.4 NA oil immersion objective (excitation, 488-nm; collection, 500–540 nm for GFP or 630–700 nm for cyanobacterial autofluorescence) or with an Olympus FLUOVIEW FV3000 (hyper-resolution) confocal laser-scanning microscope equipped with a 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). 3. Results 3.1. PG Growth during Heterocyst Differentiation in Anabaena Van-FL is a fluorescent derivative of the antibiotic vancomycin that binds to the PG precursor Lipid II in the periplasm, either bound or not to the sacculus but not involved in crosslinking, thus providing a probe for nascent PG synthesis [ 34 ]. We used Van-FL to label Anabaena filaments during heterocyst differentiation upon transfer of filaments grown in BG11 medium (containing nitrate) to BG11 0 medium lacking combined nitrogen. The differentiation process can be followed through a succession of cellular signals that include progressive loss of red fluorescence due to photosynthetic pigment rearrangement, cell elongation and enlargement, deposition of extracellular envelope layers, and finally, Life 2022,12, 1437 4 of 15 accumulation in refringent polar granules of cyanophycin resulting from N 2 fixation. In the differentiating cells, the localization of Van-FL fluorescence appeared to follow a sequence through the different stages of the differentiation process (Figure 1). Thus, at early stages (cells already enlarged but with no conspicuous change in morphology and still detectable red fluorescence), Van-FL fluorescence was detected as a homogenous peripheral signal and in foci localized in the heterocyst neck. In the neighboring vegetative cells, fluorescence was detected at the pole contiguous to the differentiating heterocyst (see the second row in Figure 1; see also [ 35 ]). Afterward, concomitant with morphological reshaping and red-fluorescence loss, the peripheral signal in the differentiating cells appeared more pronounced, especially in the polar regions. Moreover, a strong label was detected in the pole proximal to the heterocyst of the neighboring vegetative cells (see the third row in Figure 1). Later, in mature heterocysts (showing conspicuous polar granules), the peripheral signal faded, and fluorescence was more restricted to foci in the center of the heterocyst poles and the neighboring vegetative cell pole (see the fourth row in Figure 1). These observations suggest that during heterocyst differentiation, a phase of peripheral PG growth occurs at intermediate stages of differentiation (as previously noted [ 11 ]), being this a transient event. In addition, continuous activity of PG remodeling appears to be maintained at the poles of heterocysts and neighboring vegetative cell poles. 5 µm 5 µm 5 µm 5 µm S S S PPP HHH Figure 1. Van-FL staining of filaments of Anabaena sp. strain PCC 7120 during heterocyst differentiation. Filaments grown in BG11 medium were transferred to BG11 0 (lacking combined nitrogen) medium and incubated under culture conditions. After 24 (row 2) or 48 h (rows 1, 3 and 4), samples were stained with Van-FL and observed under a fluorescence microscope and photographed. Van-FL fluorescence (green), cyanobacterial autofluorescence (red), and bright-field images are shown. Cells in early stages of differentiation (S), immature heterocysts (P) and mature heterocysts exhibiting polar refringent cyanophycin granules (H) (see the text for details) are indicated. Green arrows in row 1 point to fluorescence matching the divisome complex. Life 2022,12, 1437 5 of 15 Regarding the vegetative cells of filaments incubated in BG11 0 medium, continuous peripheral Van-FL fluorescence and strong fluorescence bands in the intercellular regions, reminiscent of the pattern found in filaments growing with combined nitrogen [ 10 ], were observed (first row in Figure 1). As in the latter, in actively-growing diazotrophic filaments, the septal bands alternate in intensity, with the intensity detected in the recently formed septa being lower than in the older septa. Additionally, midcell fluorescence progressing inwards from the cell surface, matching the progressing septum under construction, could be detected in dividing cells. 3.2. Localization of MreB, MreC and MreD during Heterocyst Differentiation We studied the localization of MreB, MreC and MreD in filaments of Anabaena subjected to N-stepdown using protein fusions to GFP. For that, we used the previously generated strains CSCV6, CSCV7 and CSCV8 that express the genes sfgfp-mreB,sfgfp-mreC or sfgfp-mreD (encoding superfolder GFP fused to MreB, MreC or MreD, respectively) preceded by the native promoter of the mreBCD operon, located on an ectopic chromosomal locus, keeping an intact mreBCD operon in its native genomic location [ 10 ]. GFP fluorescence was monitored in filaments of CSCV6, CSCV7 and CSCV8 grown with nitrate and transferred to a medium lacking combined nitrogen. In the three strains, fluorescence was localized over the periphery of cells in the early stages of differentiation, as well as of immature and mature heterocysts (exhibiting complete loss of red fluorescence and conspicuous polar granules) (Figure 2), and tended to become more diffuse in older heterocysts (not shown). GFP fluorescence could also be detected frequently as foci in the heterocyst poles (Figure 2A; see CSCV8 in Figure 2B). Life2022,12,xFORPEERREVIEW5of15   reminiscentofthepatternfoundinfilamentsgrowingwithcombinednitrogen[10],were observed(firstrowinFigure1).Asinthelatter,inactively‐growingdiazotrophicfilaments,theseptalbandsalternateinintensity,withtheintensitydetectedintherecently formedseptabeinglowerthanintheoldersepta.Additionally,midcellfluorescenceprogressinginwardsfromthecellsurface,matchingtheprogressingseptumunderconstruction,couldbedetectedindividingcells. 3.2.LocalizationofMreB,MreCandMreDduringHeterocystDifferentiation WestudiedthelocalizationofMreB,MreCandMreDinfilamentsofAnabaenasubjectedtoN‐stepdownusingproteinfusionstoGFP.Forthat,weusedthepreviouslygeneratedstrainsCSCV6,CSCV7andCSCV8thatexpressthegenessfgfp‐mreB,sfgfp‐mreCor sfgfp‐mreD(encodingsuperfolderGFPfusedtoMreB,MreCorMreD,respectively)precededbythenativepromoterofthemreBCDoperon,locatedonanectopicchromosomal locus,keepinganintactmreBCDoperoninitsnativegenomiclocation[10].GFPfluorescencewasmonitoredinfilamentsofCSCV6,CSCV7andCSCV8grownwithnitrateand transferredtoamediumlackingcombinednitrogen.Inthethreestrains,fluorescencewas localizedovertheperipheryofcellsintheearlystagesofdifferentiation,aswellasof immatureandmatureheterocysts(exhibitingcompletelossofredfluorescenceandconspicuouspolargranules)(Figure2),andtendedtobecomemorediffuseinolderheterocysts(notshown).GFPfluorescencecouldalsobedetectedfrequentlyasfociintheheterocystpoles(Figure2A;seeCSCV8inFigure2B).  Figure2.LocalizationofMreB,MreCandMreDinAnabaena.StrainPCC7120andtheirderivatives CSCV6(sfGFP‐MreB),CSCV7(sfGFP‐MreC)andCSCV8(sf‐GFP‐MreD)weregrowninBG11medium,transferredtoBG110mediumandincubatedundercultureconditions.After24h,filaments werevisualizedbyconfocalmicroscopywithTCS(A)orFLUOVIEW(B)equipment.GFPfluorescence(green),cyanobacterialautofluorescence(red),mergedGFPandcyanobacterialautofluorescence,andbright‐fieldimagesareshown.Cellsinearlystagesofdifferentiation(S),immatureheterocysts(P)andmatureheterocysts(H)areindicated.GreenarrowspointtoGFPfluorescence matchingdivisomecomplexes.Magnificationisthesameforallmicrographsin(A)or(B). CSCV6 CSCV7 CSCV8 WT 10 m CSCV6 CSCV7 CSCV8 10 m A B HHH PPP SSS PPP HHH HH HHHH HHH HHH Figure 2. Localization of MreB, MreC and MreD in Anabaena. Strain PCC 7120 and their derivatives CSCV6 (sfGFP-MreB), CSCV7 (sfGFP-MreC) and CSCV8 (sf-GFP-MreD) were grown in BG11 medium, transferred to BG11 0 medium and incubated under culture conditions. After 24 h, filaments were visualized by confocal microscopy with TCS ( A ) or FLUOVIEW ( B ) equipment. GFP fluorescence (green), cyanobacterial autofluorescence (red), merged GFP and cyanobacterial autofluorescence, and bright-field images are shown. Cells in early stages of differentiation (S), immature heterocysts (P) and mature heterocysts (H) are indicated. Green arrows point to GFP fluorescence matching divisome complexes. Magnification is the same for all micrographs in (A) or (B). Life 2022,12, 1437 6 of 15 In the vegetative cells of strains CSCV6, CSCV7 and CSCV8, GFP fluorescence could also be detected in the cell periphery during active growth, and at midcell in signals progressing inwards from the periphery in the dividing cells (Figure 2). As previously noted [ 10 ], especially cells of CSCV7 and CSCV8 appeared bigger and rounder than those of the wild type. This could indicate some interference of the fusion proteins with the function of the corresponding native proteins. Nonetheless, the GFP fluorescence localization’s specificity and consistency with the signals produced by GFP-MreB (in CSCV6) support that GFP-MreC and GFP-MreD are reporting the physiological localization of MreC and MreD, respectively. 3.3. Influence of MreB, MreC and MreD in PG Growth during Heterocyst Differentiation Strains CSCV1, CSCV4 and CSCV2 contain only inactivated versions of mreB,mreC, or mreD, respectively. These mutants show strong morphological alterations, forming cells, including heterocysts, that are larger and rounder than wild-type cells [ 36 ]. Van-FL staining was performed in filaments of strains CSCV1, CSCV4 and CSCV2 grown with nitrate and incubated in the absence of combined nitrogen. Fluorescence localization was compared to the wild-type (shown in Figure 1). Regarding fluorescence distribution in differentiating cells, some heterocysts with peripheral fluorescence and septal foci could be detected in CSCV1 (see the second row in Figure 3), CSCV4 (see first and the second row in Figure 4) and CSCV2 (see the second row in Figure 5). However, instances of deformed septal fluorescence distribution or aberrantly increased labeling were also found (see the third row in Figure 3, for CSCV1, and the third row in Figure 5, for CSCV2). In the three mutants, but especially in CSCV4, few mature heterocysts with conspicuous polar granules were detected after 48 h of incubation in the absence of combined nitrogen, consistent with the previous description of retarded expression of nitrogenase activity [ 36 ]. Frequently, heterocysts with conspicuous peripheral labeling could still be detected at 48 h (see the fourth row in Figure 3for CSCV1 and Figure 5for CSCV2). CSCV1 24 h 48 h 5 µm 24 h 24 h 5 µm 5 µm 5 µm SSS S S S HHH Figure 3. Van-FL staining of Anabaena mreB mutant. Strain CSCV1 (mreB) was treated under the same conditions described for the WT (PCC 7120) in Figure 1. Van-FL fluorescence (green), cyanobacterial autofluorescence (red) and bright-field images are shown. To improve visibility, contrast/brightness is different for the image at left in row 1. The bracket indicates a cell compartment with disparate size; the blue arrow points to a tilted fluorescent band and the green arrow to fluorescence matching the divisome. Cells in the early stages of differentiation (S) and mature heterocysts (H) are indicated. Life 2022,12, 1437 7 of 15 CSCV4 24 h 48 h 24 h 5 µm 5 µm 5 µm SSS Figure 4. Van-FL staining of Anabaena mreC mutant strain CSCV4. See the legend in Figure 3for details. To improve visibility, contrast/brightness is different for the image at left in row 1. CSCV2 48 h 24 h 24 h 24 h 5 µm 5 µm PPP P P P HHH Figure 5. Van-FL staining of Anabaena mreD mutant strain CSCV2. See the legend in Figure 3for details. Immature heterocysts (P) and mature heterocysts (H) are indicated. To improve visibility, contrast/brightness differs for the images in row 1. In vegetative cells (see the first row in Figures 3–5), the three mutants showed peripheral, septal and midcell Van-FL labeling. However, the consecutive septal bands appeared frequently tilted with regard to each other or at irregular distances from each other (compare first rows in Figures 1and 3–5). Inspection of 100 to 120 septal bands of each PCC 7120, CSCV1, CSCV4 and CSCV2 incubated for 24 and 48 h in BG11 0 medium showed Life 2022,12, 1437 8 of 15 that, in the mutants, 29–40% septal bands appeared conspicuously tilted with regard to a consecutive band. In the wild type, 95–98% appeared parallel to its consecutive band (Mann–Whitney tests indicated fully significant differences, p< 0.001, for any comparison between a mutant and the WT, and no significant differences between the mutants). CSCV4 even showed some instances where the division plane appeared inverted (see row 3 in Figure 4). In addition, abundant cell debris from cell lysis was observed especially in CSCV1 and CSCV4. 3.4. Localization of FtsZ and ZipN in Mre Mutants Because heterocyst differentiation is linked to inhibition of cell division (see [ 15 ]) and because, as shown above, MreB, MreC and MreD are also localized to the divisome, we asked whether the Mre proteins affected the localization of FtsZ and ZipN during the differentiation process. FtsZ initiates the polymerization of the division ring (see [ 37 ]), and ZipN is the main tether for binding FtsZ to the cytoplasmic membrane and organizer of the divisome in Anabaena [ 38 ]. We monitored FtsZ localization using strains that express an ftsZ-gfp reporter from the P ftsZ promoter in the WT background (strain CSSC19 [ 37 ]) and in the mreB (strain CSCV20), mreC (strain CSCV21) and mreD (strain CSCV22) mutant backgrounds [ 10 ]. Upon N-stepdown, fluorescent FtsZ-rings could be detected in vegetative cells of filaments of all strains (Figure 6). However, in the mutant backgrounds, some rings were tilted compared to those of neighboring cells, and in some places, the distance between two consecutive Z-rings was irregular. In the differentiating cells described for strain CSSC19 (39), Z-rings were detected during differentiation but not in mature heterocysts. CSSC19 mostly exhibited mature heterocysts 48 h after N-stepdown, whereas in CSCV20, CSCV21 and CSCV22, cells exhibiting fluorescent rings were still abundant at that time, consistent with retardation of heterocyst differentiation. Of note is the observation of some aberrant events in CSCV20, such as the differentiation of consecutive cells (see third and fourth rows in Figure 6), which is very rare in the wild type, or the presence of a Z-ring (see the seventh row in Figure 6) or apparent septation (see the third row in Figure 6) in mature heterocysts. The effect of the lack of MreB, MreC or MreD on the localization of ZipN was studied in strains that express an sfgfp-zipN reporter gene from the P zipN promoter in the WT background (strain CSAV39 [ 39 ]) and the mreB (strain CSCV14), mreC (strain CSCV15) and mreD (strain CSCV16) mutant backgrounds [10]. Upon N-stepdown, strain CSAV39 presented midcell fluorescent bands in vegetative cells, and this signal was progressively lost in the differentiating cells, being absent in mature heterocysts (Figure 7; [ 39 ]). In CSCV14, CSCV15 and CSCV16, the midcell GFP- ZipN fluorescence was also lost in most mature heterocysts, although in some of them, some signals could still be detected (see the heterocyst in the seventh row in Figure 7 for CSCV16). In the three mutant backgrounds, but especially in CSCV15, fluorescence appeared more dispersed than in the wild-type background (strain CSAV39). Of note is the observation that in the three mutant backgrounds some heterocysts deviated from the filament longitudinal plane. Moreover, in CSCV14 and CSCV16, heterocysts with aberrant polar morphology even provoking filament breakage were observed (Figure 7). Life 2022,12, 1437 9 of 15 Life 2022, 12, x FOR PEER REVIEW 9 of 15 Figure 6. Localization of FtsZ in mreB, mreC and mreD mutants. Strains CSSC19 (ftsZ-gfpmut2 in WT background), CSCV20 (ftsZ-gfpmut2 in CSCV1 background), CSCV21 (ftsZ-gfpmut2 in CSCV4 background) and CSCV22 (ftsZ-gfpmut2 in CSCV2 background) were treated as above described for Figure 2. After 48 h, filaments were visualized by confocal microscopy with FLUOVIEW equipment. GFP fluorescence (green), merged GFP and cyanobacterial autofluorescence (red), and bright-field images are shown. To improve visibility, contrast is higher for the green image of CSCV22. Blue arrows point to tilted fluorescence bands, and blue brackets to cell compartments with disparate sizes. Immature (P) and mature heterocysts (H) are indicated. Blue arrowheads point to an apparent instance of septation in mature heterocysts; orange arrowheads to a mature heterocyst with Z-ring fluorescence; and blue asterisks to contiguous heterocysts. Magnification is the same for all micrographs. The effect of the lack of MreB, MreC or MreD on the localization of ZipN was studied in strains that express an sfgfp-zipN reporter gene from the PzipN promoter in the WT background (strain CSAV39 [39]) and the mreB (strain CSCV14), mreC (strain CSCV15) and mreD (strain CSCV16) mutant backgrounds [10]. Upon N-stepdown, strain CSAV39 presented midcell fluorescent bands in vegetative cells, and this signal was progressively lost in the differentiating cells, being absent in mature heterocysts (Figure 7; [39]). In CSCV14, CSCV15 and CSCV16, the midcell GFP- ZipN fluorescence was also lost in most mature heterocysts, although in some of them, some signals could still be detected (see the heterocyst in the seventh row in Figure 7 for CSCV16). In the three mutant backgrounds, but especially in CSCV15, fluorescence appeared more dispersed than in the wild-type background (strain CSAV39). Of note is the observation that in the three mutant backgrounds some heterocysts deviated from the filament longitudinal plane. Moreover, in CSCV14 and CSCV16, heterocysts with aberrant polar morphology even provoking filament breakage were observed (Figure 7). CSSC19 CSCV20 CSCV21 CSCV22 10 µm HHH H H H H H H PPP PPPPP P PP P HHH P P PHHH HHH PP P HHH HHH PP P PPP P P P H H H Figure 6. Localization of FtsZ in mreB,mreC and mreD mutants. Strains CSSC19 (ftsZ-gfpmut2 in WT background), CSCV20 (ftsZ-gfpmut2 in CSCV1 background), CSCV21 (ftsZ-gfpmut2 in CSCV4 background) and CSCV22 (ftsZ-gfpmut2 in CSCV2 background) were treated as above described for Figure 2. After 48 h, filaments were visualized by confocal microscopy with FLUOVIEW equipment. GFP fluorescence (green), merged GFP and cyanobacterial autofluorescence (red), and bright-field images are shown. To improve visibility, contrast is higher for the green image of CSCV22. Blue arrows point to tilted fluorescence bands, and blue brackets to cell compartments with disparate sizes. Immature (P) and mature heterocysts (H) are indicated. Blue arrowheads point to an apparent instance of septation in mature heterocysts; orange arrowheads to a mature heterocyst with Z-ring fluorescence; and blue asterisks to contiguous heterocysts. Magnification is the same for all micrographs.