Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413

Abstract

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.

FIG. 1

Promoter region of nifH2 and NtcA-dependent expression of Nif2. (A) Sequence of the region from the end of nifU2 to the start of nifH2. The transcription start site for nifH2 is indicated by a short arrow above nucleotide 4293 (numbering is based on GenBank sequence U49859 for the entire nif2 cluster) (39). (B) The transcription start site was determined by primer extension using a primer that spans nucleotides 4467 to 4490. (C) FD (✚) and MM3 (ntcA mutant) (■) cells grown with 5.0 mM NH₄Cl–10 mM TES (pH 7.2)–5.0 mM fructose were washed with AA/8, resuspended in AA/8 with 5.0 mM fructose, and incubated under anoxic conditions. Nitrogenase activity was determined by acetylene reduction assays at the times indicated.

FIG. 2

Growth and heterocyst differentiation in JE994 (⧫), FD (✚), and NF76 (●) cultures grown under anoxic, diazotrophic conditions. Cells grown with 5.0 mM NH₄Cl–10 mM TES (pH 7.2)–5.0 mM fructose were washed with AA/8, resuspended in AA/8 with 5.0 mM fructose to an OD₇₀₀ of <0.1, and incubated under anoxic conditions. Optical density (A) and heterocyst frequency (B) were determined for 5 days.

FIG. 3

Effect of exogenous ammonium on heterocyst differentiation. Cells grown with 5.0 mM NH₄Cl–10 mM TES (pH 7.2)–5.0 mM fructose were washed with AA/8, resuspended in AA/8 with 5.0 mM fructose, and incubated under anoxic conditions; 5.0 mM NH₄Cl–10 mM TES (pH 7.2) was added to aliquots of each strain at the times indicated. Incubation was continued under anoxic conditions, and heterocyst frequency was determined after 24 h. The bars labeled “none” indicate the heterocyst frequency for the control culture to which no NH₄Cl was added.

FIG. 4

Synthesis of NifH1 and NifH2 proteins in cells grown under anoxic conditions. (A) Wild-type strain FD was grown with 5.0 mM NH₄Cl–10 mM TES (pH 7.2)–5.0 mM fructose and induced at time zero under anoxic conditions as described in Materials and Methods. Samples were removed at 6 and 18 h after induction, and NifH1 and NifH2 proteins were detected on immunoblots using anti-NifH antibodies. (B) Wild-type strain FD was grown in AA/8 with 5 mM fructose for 48 h to induce heterocysts. Cells were shifted to anoxic conditions at time zero, and NifH1 and NifH2 proteins were detected on immunoblots using anti-NifH antibodies at the times indicated.

FIG. 5

In situ expression of nif2. Strain JE35 (nif2::lacZ fusion) was grown in AA/8 with 5 mM fructose for 48 h to induce heterocysts (H) and Nif1 expression. Cells were shifted to anoxic conditions, and samples were removed after 4 h and incubated with C₁₂-FDG as described in Materials and Methods. (A) Fluorescein fluorescence; (B) light micrograph. Bar = 10 μm.