Temporal and spatial regulation of the four transcription start sites of hetR from Anabaena sp. strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms nitrogen-fixing heterocysts in a periodic pattern in response to combined-nitrogen limitation in the environment. The master regulator of heterocyst differentiation, HetR, is necessary for both pattern formation and commitment of approximately every 10th cell of a filament to differentiation into a heterocyst. In this study, the individual contributions of four transcriptional start points (tsps) in regulation of transcription of hetR were assessed, and the effects of the regulatory genes patS, hetN, and patA on transcription from the tsps were determined. The tsp located at nucleotide -271 relative to the translational start site (-271 tsp) was the most tightly regulated tsp, and fluorescence from a -271 tsp-green fluorescent protein (GFP) reporter fusion was observed initially in groups of two cells and later in single cells arranged in a spatial pattern that mimicked the pattern of heterocysts that emerged. Conversely, the fluorescence from the -184 and -728/-696 tsp-GFP reporter fusions was uniform throughout filaments. Transcription from the -271 tsp was severely downregulated in a strain in which the patA gene, which encodes a positive regulator of differentiation, was deleted, and it was not detectable in strains overexpressing the genes encoding the negative regulators of differentiation, patS and hetN. In strains lacking the -271 tsp of hetR, pattern formation, the timing of commitment to differentiation, and the number of cells that differentiated into heterocysts were affected. Taken together, these results demonstrate the role of regulation of the -271 tsp of hetR in the genetic network that governs heterocyst pattern formation and differentiation.

FIG. 1.

Schematic diagrams of reporter fusions with tsp regions of the hetR promoter. (A) hetR promoter and coding region with its four transcriptional start points. (B) −728/−696 tsp-reporter fusion. (C) −728/−696(+132) tsp-reporter fusion. (D) −271 tsp-reporter fusion. (E) −184 tsp-reporter fusion. (F) −271/−184 tsp-reporter fusion. The arrows indicate tsps, and the vertical lines indicate the boundaries of sequences from the promoter region of hetR that were included in each of the fusions. The numbers are positions relative to the translational start site of hetR.

FIG. 2.

Temporal patterns of transcription from the tsps of hetR. (A) RACE products corresponding to the −728 and −696 tsps. (B) RACE products corresponding to the −271 and −184 tsps. The times (in hours) after removal of combined nitrogen are indicated below the gels.

FIG. 3.

Fluorescence micrographs (left images) and light transmission micrographs (right images) of Anabaena sp. PCC 7120 bearing (A) P_hetR-gfp in pSMC127, (B) the −728/−696 tsp-gfp fusion in pRR139, (C) the −184 tsp-gfp fusion in pRR151, (D to H) the −271 tsp-gfp fusion in pRR140, and (I) the −728/-696(+132) tsp-gfp fusion in pRR149 and (J) the patA mutant UHM101 bearing the −271 tsp-gfp fusion in pRR140. The images in panels A, B, C, G, I, and J were obtained 18 h after combined nitrogen was removed from the culture. The images in panels D, E, F, and H were obtained 4, 8, 12, and 24 h, respectively, after combined nitrogen was removed from the culture. The arrowheads indicate proheterocysts and heterocysts.

FIG. 4.

Effects of PatA, PatS, and HetN on hetR transcription. (A) RACE results. Lane 1, wild-type strain; lane 2, hetR deletion strain UHM103; lane 3, patA deletion strain UHM101; lane 4, strain 7120PN, which overexpresses hetN from the petE promoter; lane 5, wild-type strain bearing pDR211, which overexpresses patS from the petE promoter. The upper panel shows products from the −728 and −696 tsps, and the lower panel shows products from the −271 and −184 tsps. (B) Northern blot results obtained using RNA isolated after 18 h of growth in BG11₀. Lane 1, strain 7120PN, which overexpresses hetN from the petE promoter; lane 2, wild type bearing plasmid pDR211, which overexpresses patS from the petE promoter; lane 3, wild type; lane 4, hetR deletion strain UHM103; lane 5, patA deletion stain UHM101. The lower panel shows the results for methylene blue-stained RNA as loading control.

FIG. 5.

(A) β-Galactosidase activity 18 h after induction of heterocyst differentiation for the wild type bearing the −271 tsp-lacZ fusion in plasmid pRR143, the patA mutant UHM101 bearing the −271 tsp-lacZ fusion in plasmid pRR143, the wild type bearing the −728/−696(+132) tsp-lacZ fusion in plasmid pRR152, the wild type bearing the −728/−696(+132) tsp-lacZ fusion in plasmid pRR152 with 6 μM PatS-5 included in the medium, the wild type bearing the control plasmid pNC101, and the patA mutant UHM101 bearing control plasmid pNC101. WT, wild type; S-5, PatS-5; ONP, o-nitrophenol. (B) Transcription from the −271 tsp at different times after induction of heterocyst differentiation. Diamonds, β-galactosidase activity of the wild type bearing the −271 tsp-lacZ fusion; squares, β-galactosidase activity of the wild type bearing control plasmid pNC101.

FIG. 6.

Deletion of the −271 tsp region from the hetR promoter. (A and B) Photomicrographs of the −271 tsp deletion strain lacking nucleotides −295 to −268 of the hetR promoter region 24 h (A) and 48 h (B) after induction of heterocyst differentiation. (C and D) Light transmission micrograph (C) and fluorescence micrograph (D) obtained 48 h after combined nitrogen was removed from the −271 tsp deletion strain lacking nucleotides −295 to −268 of the hetR promoter region bearing P_hetR-gfp in a plasmid (pSMC127). The arrowheads indicate proheterocysts and heterocysts.