Functional Dissection of Genes Encoding DNA Polymerases Based on Conditional Mutants in the Heterocyst-Forming Cyanobacterium Anabaena PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. PCC 7120 develops N₂-fixing heterocyst cells under condition of combined-nitrogen deprivation and constitutes an excellent model for studying cell differentiation. The mechanism of heterocyst development has been extensively investigated and a network of regulating factors has been identified. A few studies have showed that the process of heterocyst differentiation relates with cell cycle events, but further investigation is still required to understand this relationship. In a previous study, we created a conditional mutant of PolI encoding gene, polA, by using a CRISPR/Cpf1 gene-editing technique. Here, we were able to create another conditional mutant of a PolIII encoding gene dnaENI using a similar strategy and subsequently confirmed the essential roles of both polA and dnaENI in DNA replication. Further investigation on the phenotype of the mutants showed that lack of PolI caused defects in chromosome segregation and cell division, while lack of DnaENI (PolIII) prevented bulk DNA synthesis, causing significant loss of DNA content. Our findings also suggested the possible existence of a SOS-response like mechanism operating in Anabaena PCC 7120. Moreover, we found that heterocyst development was differently affected in the two conditional mutants, with double heterocysts/proheterocysts found in PolI conditional mutant. We further showed that formation of such double heterocysts/proheterocysts are likely caused by the difficulty in nucleoids segregation, resulting delayed, or non-complete closure of the septum between the two daughter cells. This study uncovers a link between DNA replication process and heterocyst differentiation, paving the way for further studies on the relationship between cell cycle and cell development.

Keywords: DNA replication; SOS response; cell cycle; cyanobacteria; heterocyst.

FIGURE 1

Construction of the conditional mutant CT-dnaENI. (A) The schematic representation of the genotype of the wild type (WT) and the CT-dnaENI strain. Upstream: upstream homologous arm, Downstream: downstream homologous arm. (B) Genotype verification of CT-dnaENI strain by PCR using the primers indicated as black arrows in (A). WT was used as a control. F, R1, and R2 are short names for the oligonucleotides Pall3578F1165m, cr1_all3578F23mR, and Priboswitch2, respectively. The expected size of the PCR product amplified from the WT genome with F and R1 is indicated.

FIGURE 2

Growth of TRS-polA and CT-dnaENI under permissive and non-permissive conditions. (A) The growth curves of WT, TRS-polA, and CT-dnaENI in BG11 with or without CT (0.3 μM CuSO₄ + 1 mM TP). (B,C) The growth of TRS-polA and CT-dnaENI cultured for seven days in BG11 with indicated levels of inducers. The middle-column images are cultures from the 24-well plates. On the left and the right column are representative microscopic images of cells from the wells with indicated levels of inducers. The scale bar of 20 μm is applied for all microscopic images and is shown in the upper-left image in (B). (D) The cell size of WT, TRS-polA, and CT-dnaENI after 7-day’s cultivation in BG11 with or without inducers. 150 cells were measured for each strain and the mean cell sizes of TRS-polA and CT-dnaENI were significantly larger than that of WT according to a one-way ANOVA test (P < 0.05). ns: not significant. All experiments were done with three biological replicates.

FIGURE 3

Cell morphology, DNA contents and cell division in the conditional mutants. (A) Visualization of the nucleoids in WT, TRS-polA, and CT-dnaENI with DAPI staining. (B) Septum formation indicated by HADA labeling in WT, TRS-polA, and CT-dnaENI. In both (A) and (B), cells were cultured under non-permissive conditions for 7 days before sampled for imaging. All experiments were performed at least three times. BF: bright field. Scale bar of 10 μm is applied to all images.

FIGURE 4

The transcription levels of lexA, recA, ssb genes in WT (A), TRS-polA (B), and CT-dnaENI (C) quantified from RT-qPCR assays. -CT: without added CuSO₄ and TP in the growth medium. The transcription levels of genes were tested at the beginning of non-permissive growth (0 day), then everyday up to 4 days following transfer to non-permissive conditions, as indicated 1 day, 2 day, 3 day, and 4 day in (A). The experiment was done with three technical replicates in two biological replicates.

FIGURE 5

Heterocyst formation in DNA polymerases mutants under non-permissive conditions. As illustrated in (A), all strains were initially cultivated in BG11 with inducers to allow gene expression (permissive condition). Heterocyst induction was performed at 0 h and 48 h after the inducers were removed from the growth medium (non-permissive condition). After 24 h of induction, images of heterocyst formation under a light microscope were shown for WT (i) and TRS-polA (ii). In order to monitor heterocyst development in the mutants, transcriptional fusion of hetR promoter with gfp (P_hetR-gfp) was introduced into TRS-polA (iii) and the formation of heterocysts was recorded in both Brightfield (upper panel) and fluorescent channels (lower panel). Black or white arrows indicate single heterocyst and the red arrows indicate double heterocysts/proheterocysts. Scale bars: 10 μm. (B) DAPI staining of double heterocysts/proheterocysts in TRS-polA strain grown under non-permissive conditions. Bright field images (upper panel) and fluorescent images (bottom panel) showed presence of double heterocysts/proheterocysts at three different stages of nucleoid segregation (i, ii, and iii). Experiments were performed twice. Scale bar: 5 μm.