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An amidase is required for proper intercellular PNAS PLUS communication in the filamentous cyanobacterium Anabaena sp. PCC 7120

Zhenggao Zhenga, Amin Omairi-Nasserb, Xiying Lia, Chunxia Donga, Yan Linc, Robert Haselkornb,1, and Jindong Zhaoa,c,1

aState Key Laboratory of Protein and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People’s Republic of China; bDepartment of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637; and cChinese Academy of Sciences Key Laboratory of Phycological Research, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, People’s Republic of China

Contributed by Robert Haselkorn, December 30, 2016 (sent for review October 18, 2016; reviewed by James W. Golden and John C. Meeks) Channels that cross cell walls and connect the cytoplasm of the expression of other genes involved in heterocyst differenti- neighboring cells in multicellular cyanobacteria are pivotal for ation (14–18). The patS gene encodes a 17-aa peptide whose intercellular communication. We find that the product of the gene C-terminal pentapeptide is the inhibitor (19). The C-terminal all1140 of the filamentous cyanobacterium Anabaena sp. PCC 7120 peptide (RGSGR) prevents HetR from binding to DNA tar- is required for proper channel formation. All1140 encodes an am- gets (16), leading to the suppression of heterocyst differenti- idase that hydrolyses purified peptidoglycans. An All1140-GFP fu- ation. In the case of heterocyst pattern formation, current sion protein is located at the Z-ring in the periplasmic space during evidence supports the view that the short peptide (E)RGSGR most of the cell cycle. An all1140-null mutant (M40) was unable to moves from heterocysts and proheterocysts to neighboring grow diazotrophically, and no mature heterocysts were observed cells (19–23). in the absence of combined nitrogen. Expression of two key genes, The detailed route of the PatS peptide movement between the hetR and patS, was studied in M40 using GFP as a reporter. Upon cells has not been determined. Although it could move through nitrogen step-down, the patterned distribution of green fluores- the periplasmic space that is continuous and shared by all cells cent cells in filaments seen in the wild type were not observed in along the filaments of Anabaena 7120 (21, 24–27), we think that mutant M40. Intercellular communication in M40 was studied by the PatS peptide and other metabolites move along the filaments CELL BIOLOGY measuring fluorescence recovery after photobleaching (FRAP). through intercellular channels (26, 28–30). A recent electron Movement of calcein (622 Da) was aborted in M40, suggesting tomography (ET) study has clearly established that channels that the channels connecting the cytoplasm of neighboring cells penetrate the rigid peptidoglycan (PG) layer that separates cells are impaired in the mutant. The channels were examined with in the filaments (30). The presence of nanopore pits on the electron tomography; their diameters were nearly identical, PG septa between two cells (29) also strongly implies that 12.7 nm for the wild type and 12.4 nm for M40, suggesting that there are cytoplasmic connections between two neighboring AmiC3 is not required for channel formation. However, when the cell cells. The nanopores are located in the central areas of the wall sacculi isolated by boiling were examined by EM, the average septa. Formation of the nanopores on the septa between the sizes of the channels of the wild type and M40 were 20 nm and cellsrequiresamidasesinbothAnabaena 7120 and Nostoc 12 nm, respectively, suggesting that the channel walls of the wild punctiforme (29, 31, 32). type are expandable and that this expandability requires AmiC3. N-Acetylmuramyl-L-alanine amidases (Ami) cleave an amide bond between the N-acetylmuramic acid backbone (MurNAc) channels | peptidoglycan | cyanobacteria | intercellular communication and L-alanine of the peptidoglycan (33). There are five subgroups

he occurrence of multicellular organisms is one of the most Significance Tsignificant steps in evolution (1, 2). One of the advantages of multicellularity is that an organism can differentiate specialized The filamentous cyanobacterium Anabaena has become a cells for different functions (3, 4). In prokaryotes, there are widely studied model to determine the molecular mechanisms several independently evolved groups of multicellular organisms, involved in establishing and maintaining the pattern of het- including Actinobacteria, Myxobacteria, and the cyanobacteria erocyst differentiation in response to the removal of fixed (5). The cyanobacteria are a group of eubacteria that carry out nitrogen from the environment. Heterocysts develop from oxygenic photosynthesis. They have the most diversified mor- vegetative cells, usually spaced about 10 cells apart, convert- phology among prokaryotes, ranging from unicellular cells to ing an oxic cell capable of division into an anoxic factory for multicellular filaments with true branches (6). Anabaena sp. nitrogen fixation that does not divide. Genetic analysis to PCC 7120 (Anabaena 7120) is a filamentous cyanobacterium elucidate the mechanisms of intercellular material exchange that can form heterocysts, providing an excellent model for between heterocysts and vegetative cells is in an early phase. studying cell differentiation and pattern formation (7–9). The Here we show that an amidase is involved in the function of importance of intercellular material exchange is evident in that channels that penetrate the rigid peptidoglycan walls that heterocysts provide a micro-oxic environment for nitrogen fix- separate cells in the filaments. ation and supply nitrogenous compounds to the vegetative cells, whereas the vegetative cells perform oxygenic photosyn- Author contributions: Z.Z., A.O.-N., X.L., C.D., Y.L., R.H., and J.Z. designed research; Z.Z., thesis and supply sugars as energy and carbon skeleton to the A.O.-N., X.L., C.D., and Y.L. performed research; Z.Z., A.O.-N., X.L., C.D., Y.L., R.H., and J.Z. heterocysts (7). analyzed data; and A.O.-N., R.H., and J.Z. wrote the paper. The heterocyst pattern is dependent upon intercellular com- Reviewers: J.W.G., University of California, San Diego; and J.C.M., University of California, Davis. munication according to Turing’s activator–inhibitor model, The authors declare no conflict of interest. which requires the inhibitor to be diffusible (10, 11). Although 1To whom correspondence may be addressed. Email: [email protected] or rh01@ many genes are involved in the regulation of heterocyst forma- uchicago.edu. – tion (7 9, 12, 13), hetR and patS are most important in heterocyst This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pattern formation. HetR is a transcription factor that controls 1073/pnas.1621424114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1621424114 PNAS Early Edition | 1of8 Downloaded by guest on September 26, 2021 Results Inactivation of all1140 (AmiC3) of Anabaena sp. PCC7120. In a screen of a mutant library of Anabaena 7120 for genes that are involved in heterocyst formation, we found that an insertion mutant of all1140 was incapable of diazotrophic growth. The gene all1140 and five other genes in Anabaena 7120 (alr0092, alr0093, all4294, all4998, and all4999) encode proteins that belong to the N-ace- tylmuramoyl- L-alanine amidases based on a cyanobacterial genome database search (cyanobase; genome.microbedb.jp/cyanobase). All the amidases except All4294 have an AmiC domain. The genes alr0092 and alr0093 encode AmiC1 and AmiC2, respectively; their catalytic AmiC domain is located at the C terminus (32). The protein encoded by all1140 has an AmiC catalytic domain located at the N terminus along with two adjacent PG-binding domains at the C terminus (Fig. 1A), and the domain arrangement in All1140 is conserved in hetero- Fig. 1. Measurement of recombinant AmiC3 activity with isolated pepti- cystous cyanobacteria (Fig. S1). The AmiC domain of All1140 doglycans. (A) Schematic of the Prosite domains of AmiC3 of Anabaena is 36.1% and 33.7% identical to that of AmiC1 and AmiC2, 7120. The AmiC domain is in red, and the two PG domains are in green. (B–D) respectively, and is 32.3% identical to AmiC in E. coli. Analyses of the enzymatically digested products of PGs from S. aureus (B), All1140 protein hydrolyzes PG. Recombinant All1140 was pro- E. coli (C), and Anabaena 7120 (D) with liquid chromatography (black traces). duced in E. coli and purified (Fig. S2A). The recombinant protein The red and green traces represent control experimental analyses. was incubated with PG isolated from Staphylococcus, E. coli,and Anabaena 7120, and the hydrolyzed products were analyzed by liquid chromatography. The results (Fig. 1 B–D) show that the of these enzymes in Escherichia coli: periplasmic AmiA, AmiB, – recombinant All1140 has PG hydrolysis activity; we named this AmiC, AmiD, and cytoplasmic AmpD (34 37). In Anabaena protein “AmiC3.” AmutantofAnabaena 7120 was constructed in 7120, a mutant of amiC1 (alr0092) is unable to form hetero- which the all1140 gene was replaced by the streptomycin-resistance cysts and loses intercellular communication. Two studies of (SmR)cartridge(Fig. S2C), confirmed by Southern hybridization the adjacent homologous gene amiC2 (alr0093) reported dif- (Fig. S2B). The mutant strain is named “M40.” ferent results. Zhu et al. (38) showed that a mutant lacking Growth of the wild-type and M40 strains in BG11 (with ni- alr0093 could not form mature heterocysts, whereas Berendt trate) was measured (Fig. S3A). M40 had a longer lag time and a et al. (32) reported that a mutant lacking alr0093 showed no somewhat slower growth rate than the wild-type strain. Under observable phenotype. An amiC2 mutant of N. punctiforme these experimental conditions, the doubling time of M40 was ATCC 29133 showed irregular cell-division planes and lacked 31.9 ± 1.3 h, whereas the doubling time of the wild-type strain both cell differentiation and intercellular communication was 27.1 ± 0.7 h. M40 was not able to grow when N2 was the sole through the cytoplasm (31). Recently, the 3D structure of nitrogen source, and no heterocysts could be detected after a AmiC2 from N. punctiforme was determined, and some structural nitrogen step-down (Fig. 2A). The growth phenotype of the M40 features of the enzyme suggest that it has unique roles in cell-wall mutant was largely restored to that of the wild-type strain when remodeling (39). Here, we show that all1140, encoding a dif- M40 was complemented with a wild-type all1140 gene (C40). ferent amidase-C, is required for intercellular material exchange C40 had a doubling time of 28.8 ± 1.7 h, and it restored the in Anabaena 7120 and the differentiation of heterocysts. ability to develop heterocysts in a semiregular distribution along

Fig. 2. Light microscopic images of Anabaena 7120. (A) Bright-field (Upper) and pigment fluorescence (PF) (Lower) microscopic images of the wild-type, M40 mutant, and the C40 strains. All images were taken from cultures that had been deprived of combined nitrogen for 24 h. (B and C) GFP fluorescence (Left)or

pigment fluorescence (PF) (Right) images of Anabaena 7120 strains that had a gfp gene under control of the patS promoter (PpatS-GFP) (B)orthehetR promoter (PhetR-GFP) (C). The strains were either in a wild-type background (Upper) or in a M40 background (Lower). Images were taken from cultures that had been deprived of combined nitrogen for 12 h (B)or24h(C). Arrows indicate the positions of heterocysts (A and C) or proheterocysts (B). (Scale bars: 5 μm.)

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1621424114 Zheng et al. Downloaded by guest on September 26, 2021 the filaments (Fig. 2A). The frequency of heterocysts in the fil- PNAS PLUS aments of different strains 36 h after nitrogen starvation is shown in Fig. S3B. The heterocyst frequency of C40 was 7.1 ± 0.9%, nearly the same as that of the wild-type strain (8.7 ± 1.2%). Fusions of the gfp gene to the promoters of hetR and patS (PhetR-gfp and PpatS-gfp) were constructed to examine the expression of hetR and patS. In the wild-type background, little GFP fluorescence is observed in strains with these gfp fusions under nitrogen-replete conditions. A typical pattern of GFP- fluorescent cells can be observed in the two strains with gfp fusions in the wild-type background after nitrogen step-down (Fig. 2 B and C). On the other hand, in strain M40 the expression of hetR increased even in the presence of combined nitrogen. GFP fluorescence was observed in nearly all the cells in the M40 strain and became brighter in all cells after nitrogen step-down (Fig. 2C). However, no pattern of differentiating cells with high GFP fluorescence was observed in the M40 strain. The expression pattern of patS is shown in Fig. 2B. Little GFP fluorescence could be observed in the strain with PpatS-gfp in the presence of combined nitrogen. The cells remained dim after the removal of combined nitrogen, and no typical pattern of GFP fluorescent cells in the filaments was observed (Fig. 2B).

Subcellular Localization of AmiC3-GFP. Although there is no signal Fig. 3. Subcellular localization of AmiC3 of Anabaena 7120. (A) Localization peptide or other domain that might target AmiC3 to periplasmic of AmiC3 by cell fractionation and immunoblotting. The cytoplasmic (cyto) and outer membrane and PG (OM) fractions of the wild-type and C40G space, proteomic analysis showed that AmiC3 (All1140) existed strains and of a wild-type strain containing a gfp gene under control of the in the outer membrane fraction of Anabaena 7120 (40). In rbcL promoter (PrbcL-gfp) were obtained. The proteins in the fractions were E. coli, AmiC is located in the periplasm, and it is recruited to separated by SDS/PAGE followed by transfer to a nitrocellulose membrane. CELL BIOLOGY the Z-ring during cell division (41). To study the subcellular lo- Antibodies against GFP were used for detecting GFP and AmiC3-GFP. The cation of AmiC3, we constructed an all1140-gfp (AmiC3-GFP) molecular mass standards are shown on the left. (B) Subcellular localization fusion and transformed it into the wild-type and M40 strains. We of AmiC3-GFP. Confocal image of Anabaena 7120 C40G containing an first isolated different cell fractions and used immunoblotting to all1140-gfp fusion gene (B, 1) and its tilted (15°) image (B, 2). B, 3 and B, 4 determine which fractions contained the fusion protein. In strain are the enlarged images of the AmiC3-GFP ring in the red squares of B1 and C40G, obtained by complementing M40 with the all1140-gfp B2, respectively. (C) Light microscopic images of C40G filaments containing proheterocyst (C, 1)orheterocyst(C, 2). Panels show bright-field, GFP, pigment fusion, the majority of AmiC3-GFP was found in outer mem- fluorescence (PF), and a merged GFP and pigment fluorescence (PF+GFP) images. brane/PG fractions; a minor portion of AmiC3-GFP could be Arrowheads indicate the positions of a proheterocyst or a heterocyst. (D and E) detected in the cytoplasmic fraction (Fig. 3A and Fig. S4). In one Comparison of FtsZ-GFP rings (D, 1 and E, 1) and AmiC3-GFP rings (D, 2 and E, 2) control, a strain that expressed the gfp gene from the rbcL (ribu- in exponential growth phase (D) and in stationary phase (E). E, 3 and E, 4 are lose-1,5-bisphosphate carboxylase/oxygenase; Rubisco) promoter, enlarged images of the images in red squares of panels E, 1 and E, 2,respectively. GFP was detected only in the cytoplasmic fraction, demonstrating Arrows in D indicate the positions of newly formed septa. (Scale bars: 5 μm.) that various membranes were not contaminated with GFP. Fig. 3B shows the subcellular location of AmiC3-GFP in the filaments of these strains grown in BG11 medium (with nitrate). The GFP acetoxymethylester (AM) derivative of calcein was loaded into the fluorescence images in strain C40G (Fig. 3B) show that AmiC3- cytoplasm of Anabaena 7120 (28) where it was converted into GFP forms a ring structure at midcell positions in growing cells, hydrophilic green-fluorescent calcein (622.5 Da) (46). The possibly a result of association with the Z-rings in cell division fluorescence recovery after photobleaching (FRAP) method was (42–45). The AmiC3-GFP rings at the septa between two sepa- used to determine the capacity of the wild-type and M40 strains rated or nearly separated daughter cells were double-layered, to support calcein movement (Fig. 4). In the calcein-loaded fil- suggesting that the AmiC3-GFP rings were split by the formation aments of the wild-type strain, fluorescence of a photobleached of septa. The diameter of the AmiC3-GFP rings became smaller cell could recover with a t1/2 of 2 s (Fig. 4A and Table S1). In the as two daughter cells separated, and the ring eventually dis- calcein-loaded filaments of M40, the fluorescence of a bleached appeared from the septal regions. In the absence of combined cell did not recover during the entire observation period of 80 s. nitrogen, C40G formed heterocysts (Fig. 3C) and grew diazo- We also investigated fluorescence recovery when the cells were trophically. In nitrogen-deprived cultures of C40G, the AmiC3- loaded with fluorescent esculin, which has a molecular mass of GFP fluorescence rings were observed on both sides of a pro- 340.3 Da, smaller than calcein (Fig. 4B). Fast recovery of esculin heterocyst, whereas no AmiC3-GFP ring was seen on either side fluorescence with a t1/2 of 2 s was observed in the wild-type strain. of mature heterocysts (Fig. 3C). Because the septal ring is de- The degree of the recovery was ∼50%. In M40, a small (18%) pendent upon FtsZ, we compared the AmiC3-GFP ring with the recovery of esculin fluorescence was observed with a recovery t1/2 FtsZ-GFP ring. Although both rings are located in the septal of 9 s (Table S2). regions (Fig. 3D), the AmiC3 ring persisted longer in cell cycles. The lack of intercellular movement of calcein in M40 was Another difference was observed in the stationary phase: FtsZ- investigated further using EM. In transmission EM (TEM), im- GFP was located mostly in the cytoplasm, whereas AmiC3-GFP ages of thin sections after chemical fixation show that the was located mostly in the periplasmic space (Fig. 3E). channels that physically connect two vegetative cells are present in both the wild-type and M40 strains (Fig. 5 A and B). Con- Intercellular Molecule Movement and Septal Nanopores (Channels). nections between a vegetative cell and heterocyst were observed To determine whether AmiC3 is required for intercellular also (Fig. S5). Next, high-pressure freezing combined with ET communication, we investigated whether small molecules are was used to investigate the channels of the wild-type and M40 able to move between the cells in Anabaena 7120. A nonfluorescent strains; the results are shown in Fig. 5 C and D. The septal

Zheng et al. PNAS Early Edition | 3of8 Downloaded by guest on September 26, 2021 Fig. 4. FRAP of Anabaena 7120 strains. The wild-type and mutant M40 strains were loaded with calcein (A, 1) or esculin (B, 1) before photobleaching. As indicated by arrowheads, a cell was photobleached, and its fluorescence was monitored continuously. Images before photobleaching (Pre) and at 0, 4, 20, and 80 s after photobleaching are shown. A, 2 and B, 2 show the kinetics of fluorescence intensities as a function of the recovery time of filaments loaded with calcein and esculin, respectively. (Scale bars: 5 μm.)

nanopores that are required for the formation of channels be- S7) predicts that the catalytic domain of AmiC3 is very similar to tween cells are present as opaque areas of PG wall in perpen- that of the N. punctiforme AmiC2 (39). Fractionation of cellular dicular sections of the septa. Our measurements show an average proteins revealed that the AmiC-GFP fusion protein is located in diameter of 12.7 nm and 12.4 nm for the wild-type and M40 the periplasmic space (Fig. 3A), even though there is no signal strains, respectively. The channels could be observed best when peptide in the primary sequence of AmiC3. Confocal microscopy the images are rotated 90° (Fig. 5 C, 2 and D, 2). The septal demonstrated that AmiC3-GFP is associated with the septal nanopores were examined further with isolated cell-wall sacculi rings (Fig. 3B). A similar situation was found for the AmiC from Anabaena 7120 strains according to Lehner et al. (29); protein of E. coli, which has no signal peptide but is located in results are shown in Fig. 5 C and D. Although arrays of nano- the periplasmic space (41). In Anabaena 7120, AmiC3 is a per- pores are observed in the septa of both the wild-type and M40 sistent component of the septal ring: The association of AmiC3- strains (Fig. 5 E, 2 and F, 2), the sizes of nanopores in the iso- GFP with the septal ring can be observed for nearly the entire lated sacculi of these two strains are quite different: The average period of septum formation. The behavior of the AmiC3-GFP diameter of the nanopores in the M40 strain is 11.7 nm, similar ring during the cell cycle (Fig. 3B) indicates that AmiC3 is tightly to that obtained by ET, whereas the average diameter of the associated with inward-growing cell septa even after the disas- nanopores of the wild-type strain is 20.1 nm, significantly larger sembly of the FtsZ-ring (Fig. 3D). It also shows that AmiC3 in than that obtained by ET. Anabaena 7120 performs its function in a restricted area: the newly formed PG layer of the cell septa. The M40mutant lacking FRAP in Nonheterocystous Strains of Cyanobacteria. In a survey of the all1140 gene encoding AmiC3 showed no fluorescence the ability to recover fluorescence after photobleaching in fila- recovery after a cell loaded with calcein was photobleached mentous cyanobacteria, we found two nonheterocystous strains (Fig. 4), indicating that cellular communication is impaired in that showed no calcein fluorescence recovery when a cell was the mutant. bleached: Phormidium sp. and Oscillatoria sp. (Table 1 and Fig. Because the cyanobacterial cells have a rigid PG layer in cell S6). Septal nanopores were examined with the sacculi isolated walls, the formation of the channels for material exchange be- from these two strains and a Microcoleus sp. strain, which showed tween cells would require the formation of nanopores in septa, fast fluorescence recovery (Fig. S6). The average diameters of and therefore it is not surprising that amidases are involved in the septal nanopores of Phormidium sp. and Microcoleus sp. were these processes. An early study with freeze-fracture EM showed 12.5 nm (Fig. 6A) and 20.0 nm (Fig. 6B), respectively (Table 1). that some filamentous cyanobacteria had channels between the The nanopore in Oscillatoria sp. was unique in that it had only cells (47). These channels were called “microplasmodesmata,” one central nanopore on a septum, and the diameter of the but their relationship to plant connections was not established in nanopore was 23.3 nm (Fig. 6C). terms of detailed structure or function. Several recent studies demonstrated that nanopores or channels are indeed present in Discussion the septa of heterocystous cyanobacteria (30). These studies Like AmiC1 and AmiC2, AmiC3 of Anabaena 7120 had an agreed that there is an array of nanopores in the septum, but the AmiC domain, and its amidase activity was demonstrated by its reported size of the nanopore differed in these studies. A diameter ability to hydrolyze PG isolated from E. coli, Staphylococcus of 12 nm was found when ET was used to study the channels aureus, and Anabaena 7120 (Fig. 1). Computer modeling (Fig. crossing the septum in Anabaena 7120 (30). When sacculi of

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Fig. 5. Analyses of septal nanopores and connections by EM. (A and B) TEM images of the wild-type strain (A, 1), its enlarged image (A, 2), the M40 mutant (B, 1), and its enlarged image (B, 2). (C–F) C, 1 and D,1 are ET images of samples of the wild-type and M40 strains, respectively, frozen at high pressure. C, 2 and D, 2 correspond to a 90° rotation around the y axis of the septa of C, 1 and D, 1, respectively. C, 3 and D, 3 are the enlarged images of middle parts of C, 1 and D, 1, respectively. E, 1 and F, 1 are the images of isolated sacculi from the wild-type and M40 strains, respectively. E, 2 and F, 2 are the images of septa with nanopores from the wild type and M40, respectively. All tomographic images are composed of 10 superimposed 2.2-nm tomographic slices. (Scale bars: 100 nm in C, 2; C, 3; D, 2; and D, 3; 500 nm in all other panels.)

N. punctiforme were isolated by boiling and were examined by AmiC2 of N. punctiforme. More importantly, we found that the EM, the observed diameter of septal nanopores was 20 nm (29). septal walls of the wild-type and M40 strains respond differ- Because the size of nanopores is important for their function, we ently to the treatment during sacculi isolation. The septal wall compared the two methods. We found that the different nanopore of the wild-type strain was expandable, and the expandability sizes reported by various laboratories do exist in the wild-type was dependent upon AmiC3. Apparently, the ability to expand Anabaena 7120 when the two different methods mentioned above is important for proper channel formation to allow molecules were used to observe the nanopores. However, when M40 lacking with certain mass to move between the cells. That some fluo- AmiC3 was studied, very similar sizes of nanopores were observed rescence recovery was observed when the cells of the M40 with the same two methods (Fig. 5). These results indicate that the strain were loaded with esculin (Fig. 4) supports this sugges- function of AmiC3 is not directly involved in pore drilling as is tion. It is also interesting that Phormidium sp. (Fig. 6) and

Table 1. Measurement of diameters of septal nanopores and fluorescence recovery times of filamentous cyanobacteria

Species Diameter of nanopores, nm Recovery time t1/2,s

Anabaena sp.7120 WT 12.7 ± 1.3 (ET)* - Anabaena sp.7120 M40 12.4 ± 0.7 (ET)* - Anabaena sp.7120 WT 20.07 ± 0.42 (sacculi)† 2.17 ± 1.14 † Anabaena sp.7120 M40 11.66 ± 0.53 (sacculi) >80 † Anabaena cylindrica 25.55 ± 0.46 2.06 ± 0.93 Microcoleus vaginatus 19.95 ± 0.81† 3.13 ± 2.67 Oscillatoria lutea 23.32 ± 0.52† >80 † Phormidium foveolarum 12.49 ± 0.45 >80

Two methods were used in measurement of the nanopore diameters: observation of isolated sacculi with TEM and ET of cryopreserved filaments. For determination of fluorescence recovery time, filaments were loaded with calcein, and fluorescence recovery was measured after a cell was photobleached. The strains used were obtained from the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences, Wuhan, China. *Data from ET images of samples frozen under high pressure. †Data from TEM images of purified PG sacculi.

Zheng et al. PNAS Early Edition | 5of8 Downloaded by guest on September 26, 2021 expression of other downstream genes for heterocyst formation. However, even though hetR was up-regulated in the absence of combined nitrogen in the M40 strain (Fig. 2), the patS gene, which is a downstream gene regulated by hetR,wasnotup- regulated, and no morphological differentiation was observed (Fig. 2). Overexpression of hetR, which usually induces multiple contiguous heterocyst formation (15), did not induce the formation of heterocysts (Fig. S3) in the M40 strain. Because the molecular mass of the short C-terminal peptide of PatS [(E) RGSGR] is similar to that of calcein, its movement from cell to cell could be impeded in the M40 strain. How do we explain the inability of the mutant strain M40, which fails to make the enzyme AmiC3, to differentiate heterocysts? Differentiation requires the activity of HetR (14). The hetR gene is transcribed in response to a regulatory cascade that begins with deprivation of fixed nitrogen, in particular a reduction in the ratio of glutamine to 2-oxoglutarate. The latter activates NtcA, which activates transcription of nrrA (49), which in turn activates transcription of hetR. Thus, HetR is produced in all the cells of the filament of Anabaena 7120.Therefore,onewould expect that all cells should differentiate. However, one gene that is immediately activated by HetR is patS; its product, the protein PatS, whose C-terminal hexapeptide, ERGSGR, is released by proteolysis, binds to HetR, and causes it to dissociate from target DNA (8, 16, 19). HetR freed from DNA is destroyed rapidly by protease. Therefore, the critical gradient that determines the pattern of differentiated cells should be a gradient of PatS peptide whose concentration is highest next to a heterocyst and lowest halfway between two heterocysts (23, 28). The full PatS protein must be made in the heterocyst, where there is abundant active HetR. Logic suggests that the protease that generates the inhibitory peptide from PatS is located in or near the channels that connect heterocysts to vegetative cells. Our FRAP experiments indicate that the channels through the peptidoglycan layer are a correct size to transport the PatS peptide. To complete this model, we must conclude that the channels made in the mutant M40 strain are defective in PatS processing and transport, so inhibitory PatS levels remain in all cells. Without export of PatS there can be no functional HetR and no heterocysts. Experimental Procedures Gene Inactivation and Complementation. All enzymes were purchased from Promega and used according to instructions. To construct amiC3 (all1140) mutants, a DNA fragment containing amiC3 was amplified by the PCR with primers P3 and P4, using total genomic DNA as template. All primers used in PCR are listed in Table S3; all PCR products were confirmed by sequencing. The PCR-generated fragment was cloned in pGEM-T vector (Promega) to Fig. 6. Analyses of septal nanopores from filamentous nonheterocystous generate plasmid pTv-amiC3. The plasmid was inversely amplified by PCR cyanobacteria by EMy. (A–C) Images from Phormidium foveolarum (A), with primers P5 and P6. The generated fragment was ligated with a blunt- Microcoleus vaginatus (B), and Oscillatoria lutea var. contorta (C), respectively. ended SmR cartridge encoding resistance to streptomycin. The resulting (A, 1, B, 1,andC, 1)Isolatedcellwallsacculi.(A, 2, B, 2,andC, 2)EMimagesof plasmid pAmiC3-Sm was digested with BglII and PstI, and the recovered septa with nanopores. (A, 3, B, 3,andC, 3) Enlarged images of a nanopore in fragment then was cloned into pRL277 (50) for transformation of Anabaena A, 2, B, 2,andC, 2, respectively. (Scale bars: 500 nm in A and B;20nminC.) 7120 by conjugation. Segregation of the mutant amiC3 was confirmed by Southern hybridization as described by Huang et al. (16). Total genomic DNA of Anabaena 7120 was isolated with the E.Z.N.A. Plant DNA Miniprep Kit Microcoleus sp. have 12-nm and 20-nm nanopores on septa of (Omega Biotek) and then was digested with ClaI and EcoRI. The digested isolated sacculi, respectively. The former displays no calcein fragments were separated on a 1.0% agarose gel before transfer onto ni- fluorescence recovery, but the latter does. In addition to trocellulose paper for hybridization. The DNA probe was synthesized by random primer extension using the template amplified with primers P7 and nanopore size, the number of nanopores on septa is also im- P8. The mutant with all1140 mutation was named M40. For complementa- portant. In Oscillatoria sp., which has just one nanopore at the tion of M40, the amiC3 gene plus a 600-bp upstream section were amplified center of the septum, no calcein fluorescence recovery was by PCR using primers P9 and P10 followed by insertion into a chromosome detected even though the nanopore has a diameter of 23 nm docking site according to Liu (51). (Fig. 6). To construct PhetR-gfp and PpatS-gfp as reporters of hetR and patS ex- The ability of a vegetative cell to differentiate and form het- pression, the gfp gene was first amplified with primers P12 and P14, and the erocysts for nitrogen fixation represents an advanced feature of fragment was digested with SalI and EcoRI. It then was ligated to pAM505 to generate plasmid pAM505-gfp with the same enzymes. The hetR and the multicellularity in prokaryotes. Among many genes that are in- patS promoters (14, 19) were amplified by primers P15/P16 and P17/P18, volved in heterocyst formation, hetR together with patS plays a respectively, and the generated fragments were cloned into the BamHI

central role in the process. The initiation of heterocyst differentiation and SalI sites of pAM505-gfp, generating pAM505-PhetR-gfp and pAM505- requires up-regulation of hetR (48), which in turn regulates the PpatS-gfp. They were transformed into the wild-type and the M40 strains.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1621424114 Zheng et al. Downloaded by guest on September 26, 2021 To express the hetR gene from the petE promoter, the petE promoter (15) Probes). The suspension was incubated at 30 °C in the dark for 90 min, and PNAS PLUS amplified with P19 and P20 was cloned into pAM505, generating plasmid cells were washed three times with fresh BG11 medium. The suspension was

pAM505-PpetE. The hetR gene amplified with P21 and P22 was cloned into incubated for another 90 min in the dark before imaging. the SalI and EcoRI sites of pAM505-PpetE, and the resultant plasmid pAM505- For FRAP, the suspensions were spotted onto agar-coated glass slides PpetE-hetR was transformed into the wild-type and M40 strains separately. [1.5% (wt/vol) Bacto-Agar with growth medium]. All measurements were Confocal images of Anabaena 7120 were obtained with a Zeiss LSM 710 NLO carried out at room temperature (∼25 °C). FRAP experiments were performed DuoScan System confocal microscope using a Plan-Apochromat 63×/1.40 iil on an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer) with a differential interference contrast (DIC) M27 objective. Photosynthetic pig- 100×/1.4 NA oil-immersion objective and a solid state 50-mW 488-nm laser ment fluorescence images (excitation: 561 nm; emission detection: 600–650 nm) line. Calcein fluorescence was imaged with 488-nm excitation and 525/50-nm and GFP fluorescence images (excitation: 488 nm; emission detection: 495–540 nm) emission filters. After three prebleach images were recorded using10% laser were recorded. power, a region of interest was bleached used a single iteration with the 488-nm line operating at 100% laser power, which lasted 50–80 ms depending Localization of AmiC3 and FtsZ. To localize AmiC3, a gfp gene was fused to the on the bleach region size. Fluorescence recovery was monitored at low laser C-terminal part of amiC3 as follows. A gfp gene was amplified with PCR by intensity (10% of a 50-nW laser) over 80 s at 0.5-s intervals. FRAP data were primers P11 and P12 using the plasmid pAM1951 (19) as template before it analyzed using Velocity software. was ligated to pAM505 to generate plasmid pAM505-gfp-2. The PamiC3- amiC3 was amplified using primers P9 and P13 and was cloned into the Esculin Labeling and FRAP. For esculin labeling (53), cell cultures were grown BamHI and SalI sites of pAM505-gfp-2. The resultant plasmid, pAM505- on BG11 medium until the OD730 was 0.6. Then 0.5 mL of culture was har- PamiC3-amiC3-gfp, was transformed into the M40 strain by conjugation to vested by gentle centrifugation and was washed three times with fresh BG11 generate the C40G strain. FtsZ was localized according to Sakr et al. (43). medium. The cell pellet was resuspended in 0.5 mL fresh BG11 and mixed First, the ftsZ gene was amplified using primers P23 and P24 by PCR with with 15 μL of saturated (∼5 mM) aqueous esculin hydrate solution (Sigma- Anabaena PCC 7120 genomic DNA as a template and was cloned into the Aldrich). The suspension was incubated at 30 °C in the dark for 30 min, and SacI and SalI sites of pAM505-PpetE to generate the intermediate vector, cells were washed three times with fresh BG11 medium. The suspension was pAM505-PpetE-ftsZ. Second, the gfp gene amplified with PCR by primers P11 incubated for another 15 min in the dark before imaging. and P12 was digested with SalI and EcoRI and then ligated to pAM505-P - petE For FRAP, the suspensions were spotted onto agar-coated glass slides ftsZ; the resultant plasmid pAM505-P -ftsZ-gfp was transformed into wild- petE [1.5% (wt/vol) Bacto-Agar with growth medium]. FRAP experiments were type Anabaena PCC 7120. performed as described above for calcein, except that the bleach used a The cell suspensions were spotted onto agar-coated glass slides [1.5% (wt/vol) 430-nm line and emission was recorded at 474/501 nm. Bacto-Agar in growth medium]. All measurements were carried out at room temperature (∼25 °C). 3D-SIM images were obtained on an N-SIM imaging

EM and Cryo-EM. Peptidoglycan sacculi were prepared by the method of de CELL BIOLOGY system (Nikon) equipped with a 100×/1.49 NA oil-immersion objective (Nikon) and four laser beams (488 and 561 nm). Image stacks with an interval Pedro et al. (54) and Lehner et al. (29). EM of purified PG sacculi was done of 0.12/0.24/0.48 μm were acquired and computationally reconstructed to according to Priyadarshini et al. (55) and Lehner et al. (31). The samples of generate superresolution optical serial sections with twofold extended reso- ultrathin sections were obtained according to Fiedler et al. (56) and were lution in both x,y and z directions. The reconstructed images were processed observed with a JEM-1010 electron microscope (JEOL). Cryopreservation of further for maximum-intensity projections and 3D rendering with NIS- Anabaena 7120 strains and EM tomography were performed as described by Elements AR 4.20.00 (Nikon). Omairi-Nasser et al. (30). To determine which cellular compartment contains AmiC3, cell fraction- The experimental procedures of strains and culture conditions, protein ation was performed with the strain C40G and a strain expressing gfp purification, and enzyme assays are provided in SI Experimental Procedures. according to Bölter et al. (52) and Moslavac et al. (40). Proteins in different fractions were analyzed by immunoblotting using antibodies against GFP. ACKNOWLEDGMENTS. We thank the Core Facilities at the College of Life Sciences, Peking University for assistance with confocal microscopy and TEM; H. Lu, C. San, and Y. Hu of the College of Life Sciences, Peking University for Calcein Labeling and FRAP. Calcein labeling was done according to Mullineaux help with the EM work; and Jotham Austin II for assistance with cryo-EM et al. (28) and Lehner et al. (31). For calcein labeling, cultures were grown measurements performed using the facility in the Biological Science Division until the OD730 was 0.6. Then 0.5 mL of culture was harvested by gentle at the University of Chicago. This research is supported by the National centrifugation and was washed three times with fresh BG11 medium (6). The Science Foundation of China Grant 30230040, the Chinese Academy of Sci- pellet was resuspended in 0.5 mL fresh BG11 medium and was mixed with ences Grant QYZDY-SSW-SMC004, and The Ministry of Science and Technol- 20 μL of calcein-Am (1 mg/mL in dimethylsulphoxide; Invitrogen Molecular ogy of China Grant 01CB108903.

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