Inhibition of cell division suppresses heterocyst development in Anabaena sp. strain PCC 7120

Abstract

When the filamentous cyanobacterium Anabaena PCC 7120 is exposed to combined nitrogen starvation, 5 to 10% of the cells along each filament at semiregular intervals differentiate into heterocysts specialized in nitrogen fixation. Heterocysts are terminally differentiated cells in which the major cell division protein FtsZ is undetectable. In this report, we provide molecular evidence indicating that cell division is necessary for heterocyst development. FtsZ, which is translationally fused to the green fluorescent protein (GFP) as a reporter, is found to form a ring structure at the mid-cell position. SulA from Escherichia coli inhibits the GTPase activity of FtsZ in vitro and prevents the formation of FtsZ rings when expressed in Anabaena PCC 7120. The expression of sulA arrests cell division and suppresses heterocyst differentiation completely. The antibiotic aztreonam, which is targeted to the FtsI protein necessary for septum formation, has similar effects on both cell division and heterocyst differentiation, although in this case, the FtsZ ring is still formed. Therefore, heterocyst differentiation is coupled to cell division but independent of the formation of the FtsZ ring. Consistently, once the inhibitory pressure of cell division is removed, cell division should take place first before heterocyst differentiation resumes at a normal frequency. The arrest of cell division does not affect the accumulation of 2-oxoglutarate, which triggers heterocyst differentiation. Consistently, a nonmetabolizable analogue of 2-oxoglutarate does not rescue the failure of heterocyst differentiation when cell division is blocked. These results suggest that the control of heterocyst differentiation by cell division is independent of the 2-oxoglutarate signal.

FIG. 1.

Subcellular localization of FtsZ_Ana. The strain containing the plasmid pSS10c bearing an ftsZ-gfp fusion was grown in BG11 for 48 h and visualized under bright light (A) or by epifluorescence excited by light of 465 nm (B). (C) The same results as in panel B are shown but observed with a confocal microscope to show the ring structure of FtsZ in one cell. Bars, 5 μm.

FIG. 2.

Effect of sulA expression on the formation of FtsZ rings. The strain carrying two compatible plasmids pSS10c (with an ftsZ-gfp fusion) and pSS14 (with the sulA gene) were grown in BG11 without (A and B) or with (C and D) 0.2 μM CuCl₂. The same filaments were pictured under bright light (A and C) or under epifluorescence excited by light of 465 nm (B and D). (E and F) Controls representing the wild-type strain grown in BG11 with 0.2 μM CuCl₂, pictured under the microscope under the same conditions. The background fluorescence is due to the presence of photosynthetic pigments. Scale bars, 5 μm.

FIG. 3.

Effect of SulA on the GTPase activity of FtsZ_Ana. The GTPase activity of FtsZ_Ana was assayed by a coupled GTP-regenerating enzyme system (29). A total of 0.9 nmol of FtsZ was preincubated without or with 0.225, 0.45, or 0.9 nmol of MBP-SulA, and the amount of hydrolyzed GTP was determined.

FIG. 4.

Inhibition of FtsZ ring formation leads to cell elongation and suppresses heterocyst differentiation. The wild-type strain (A and C) and a strain bearing pSS5b containing the sulA gene (B and D) were grown for 48 h in BG11 containing 0.2 μM CuCl₂ (A and B) and then transferred into BG11₀ with the same amount of CuCl₂. At 24 h after the transfer, heterocyst differentiation (indicated by arrows) was visualized (C and D). Bars, 5 μm.

FIG. 5.

Oxygen evolution of filaments of the wild-type strain, the strain overexpressing sulA, or the wild-type strain treated with the antibiotic aztreonam. The rate of oxygen evolution was measured as previously described (22) using cell suspensions containing to 7 to 10 μg chlorophyll/ml. The rate of oxygen evolution is calculated relative to that obtained just before the depletion of combined nitrogen (time zero). Open bars, wild-type filaments incubated in nitrate-containing BG11 and then transferred into BG11₀ without nitrate; black bars, wild-type filaments treated with aztreonam for 2 days in BG11 and then transferred into BG11₀ with aztreonam; dotted bars, wild-type filaments overexpressing sulA cultured in BG11 containing 0.2 μM copper as an inducer of sulA expression and then transferred into BG11₀ containing the same concentration of copper.

FIG. 6.

Effect of aztreonam on cell division and FtsZ localization. Wild-type Anabaena grown for 48 h in BG11 (A) or BG11 with 40 μg · ml⁻¹ of aztreonam (B) was visualized by bright-field light microscopy. A strain carrying the plasmid pSS10c (with an ftsZ-gfp fusion) was treated with aztreonam for 2 (B) or 4 (C) days and visualized by bright-field light microscopy (B and C) or epifluorescence (D) to show the effect on FtsZ ring formation. Bars, 5 μm.

FIG. 7.

Reinitiation of cell division after removal of aztreonam. (A) A cell population in the exponential phase of growth. (B and C) Cells treated with aztreonam for 48 h and then transferred into a medium without aztreonam for 24 h (B) or 48 h (C) are shown. The lengths of 400 cells for each experiment shown in panels A to C were measured and classified into fractions corresponding to those 2.1 to 2.5 μm in length (labeled as 2.3), those 2.6 to 2.9 μm in length (labeled as 2.7), those 2.9 to 3.5 μm in length (labeled as 3.2), those 3.6 to 4.4 μm in length (labeled as 4), and 4.5 to 5.2 μm in length (labeled as 4.8).

FIG. 8.

Induction of heterocyst differentiation in the presence and following removal of aztreonam. A culture was treated with aztreonam for 48 h in the presence of nitrate as nitrogen source (A), then aztreonam and nitrate were both removed to induce heterocyst differentiation, and heterocysts were observed 24 h (B) or 48 h (C) after induction. In the control, heterocysts were induced from a culture untreated with aztreonam and observed for 24 h (D) or 48 h (E) after induction. (B and D) Filaments were incubated with Alcian blue, which specifically stains the polysaccharide layer of heterocysts. Some cells, as indicated by arrowheads (B), are still irregular in size; heterocysts are indicated by arrows (C to E).

FIG. 9.

The levels of 2-oxoglutarate (2OG) were not affected by inhibition of cell division. The wild-type strain (dotted bars), the strain bearing pSS5b cultured in the presence of copper to induce the expression of sulA for 2 days (striped bars), and the wild-type strain treated with aztreonam for 2 days (black bars) were cultured in the presence of nitrate (time zero) and then shifted to a medium without nitrate for 1 or 2 h. The levels of 2OG were then measured. The results were the means of two experiments.