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

Abstract

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.

Keywords: coiled-coil-rich proteins; divisome-dependent localization; filamentous cyanobacteria; septal peptidoglycan nanopores; septal proteins.

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 2

Localization of SepT-GFP in Anabaena PCC 7120 and BS1 mutant. Strains Anabaena WT, CSCV25 (expressing SepT-GFP in the wild-type background), and CSCV26 (expressing SepT-GFP in BS1 background) were grown in and transferred to BG11 medium at a cell density corresponding to 0.5 µg Chl mL⁻¹ and incubated under culture conditions. After 24 hours, filaments were observed by confocal microscopy with a Fluoview equipment. GFP fluorescence (green) and merged GFP and cyanobacterial autofluorescence (magenta) images are shown. Yellow arrows point to GFP fluorescence matching the divisome. Magnification is the same for all micrographs.

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

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 NaNO₃ ⁻) or BG11₀ (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 OD₇₅₀ (At) was measured in aliquots of each culture. The values of two independent cultures of each condition (one culture of CSCV9 under N₂) were represented and adjusted to sequential linear functions. A0 represents the OD₇₅₀ 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 OD₇₅₀ 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

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 BG11₀, at a cell density corresponding to 0.5 µg Chl mL⁻¹, and incubated under culture conditions. (A) After 48 hours, filaments from BG11₀ 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

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 BG11₀ (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 bright-field 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 7

Localization of MreB, MreC, and MreD in the absence of SepT. Strains CSCV11 (sepT::C.K1, sfgfp-mreB), CSCV12 (sepT::C.K1, sfgfp-mreC), CSCV13 (sepT::C.K1, sfgfp-mreD), and reference strains Anabaena WT, CSCV9 (sepT::C.K1), CSCV6 (sfgfp-mreB), CSCV7 (sfgfp-mreC), and CSCV8 (sfgfp-mreD) were grown in BG11 medium and transferred to BG11₀ + NH₄ ⁺ (A) or BG11₀ medium (B), adjusted to a cell density corresponding to 0.5 µg Chl mL⁻¹. After 24 hours, aliquots of filaments were observed under a TCS confocal microscope and photographed. GFP fluorescence (green), cyanobacterial autofluorescence (magenta), and bright-field images are shown. Yellow arrows point to heterocysts. Magnification is the same for all micrographs.

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). 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).