Cyanobacterial cell lineage analysis of the spatiotemporal hetR expression profile during heterocyst pattern formation in Anabaena sp. PCC 7120

Abstract

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.

Figure 1. Heterocyst differentiation in microelectromechanical system-assisted liquid microchambers.

Spatiotemporal dynamics in morphological changes (BF, bright field), hetR promoter (PhetR) activity monitored with a transcriptional gfp fusion reporter (hetR), and phycobilisome fluorescence (Pbs) in an individual Anabaena filament at the indicated times after nitrogen step-down. The dashed square at the top indicates the space of a microchamber. The bottom panel (taken at 70 h) shows a magnification of part of the filament.

Figure 2. Cell lineage of an individual Anabaena filament.

(A) The cell lineage analysis representing spatiotemporal profiles of cell division (branches) and heterocyst differentiation (red) in the bacterial filament shown in Figure 1. For a magnified view, see Figure S2A. The horizontal axis shows time (h) after nitrogen step-down. (B, C) Magnification of a part of the Anabaena cell lineage shown in the blue square in panel A (B) and the same lineage superimposed with the spatiotemporal dynamics of PhetR::gfp signals (C). For a full scale image, see Figure S3A. Arrowheads indicate some cells showing different hetR expression dynamics (see text). (D) Micrographs of PhetR::gfp expression patterns from the corresponding part of the bacterial filament. (E) Cells that differentiated into heterocysts without cell divisions (numbers 12 and 39). These are parts of the complete cell lineage shown in Figure S2D.

Figure 3. Initial condition-independent selection of leading heterocyst positions and dynamic hetR expression profiles.

(A) The distributions of cells at the beginning of nitrogen step-down, which generated progenies differentiating into leading heterocysts, are indicated by open circles. The other cells are shown by filled circles. (B) Scatter plot of PhetR::gfp and phycobilisome fluorescence signals for cells whose daughter cells differentiated into leading or very early heterocysts (open circle) and the other cells (filled circle) at the beginning of nitrogen step-down. No statistically significant difference was found between the two cell groups for both fluorescence signals using two-sided Student's t-test (five filaments with 92 cells). (C, D) Transition PhetR::gfp (C) and phycobilisome (D) fluorescence signals in an individual Anabaena filament during the course of heterocyst formation from 42–72 h after nitrogen step-down. Cells were categorized into four groups whose progenies (or themselves) differentiated into heterocyst(s): (1) at 62 h after nitrogen step-down (the leading heterocyst, red); (2) at 63–65 h (during the transition state, magenta); (3) at 66–72 h (after establishment of regular patterns, orange) and (4) the remaining vegetative cells at 72 h (green). For data shown in panel C and D, fluorescence intensity was normalized globally so that the mean value per cell was 1.0.