cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

Abstract

Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of these genes is cyabrB1 (also known as calA in Anabaena sp. strain PCC 7120), encoding a transcriptional regulator. In the present study, we investigated the function of calA/cyabrB1 in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method that we recently utilized for the photosynthetic production of a useful chemical in this strain. Conditional knockdown of calA/cyabrB1 in the presence of nitrate resulted in the formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were upregulated by calA/cyabrB1 knockdown in the presence of combined nitrogen sources. These genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by calA/cyabrB1 knockdown. hetP and hepA were repressed by direct binding of CalA/cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the overexpression of calA/cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of calB/cyabrB2 (a paralogue gene of calA/cyabrB1), in addition to knockdown of calA/cyabrB1, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and CalA/cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen stepdown. We concluded that cyAbrB proteins are essential safety devices that inhibit heterocyst differentiation.IMPORTANCE Spore formation in Bacillus subtilis and Streptomyces has been extensively studied as models of prokaryotic nonterminal cell differentiation. In these organisms, many cells/hyphae differentiate simultaneously, which is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. strain PCC 7120 is unique because it is terminal, and only 5 to 10% of vegetative cells differentiate into heterocysts. In this study, we identified CalA/cyAbrB1 as a repressor of two genes that are essential for heterocyst formation independently of HetR, a master activator for heterocyst differentiation. This finding is reasonable for unique cell differentiation of Anabaena because CalA/cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is overexpressed could differentiate into heterocysts.

Keywords: CRISPR interference; CalA/cyAbrB1; essential genes; hetP; heterocyst.

FIG 1

Promotion of heterocyst formation by knockdown of calA/cyabrB1 in the presence of nitrate. A control strain lacking sgRNA (C100) and a calA/cyabrB1 knockdown strain (C104) were cultured in nitrate-containing medium in the absence or presence of the inducer aTc (50 ng/ml). Cultures were bubbled with air containing 5% (vol/vol) CO₂. (A) Formation of heterocysts by calA/cyabrB1 knockdown in the presence of nitrate. Images were photographed after 48 h of cultivation. Arrowheads indicate heterocysts. (B) Confirmation of CalA/cyAbrB1 knockdown. After 48 h of cultivation, total protein was extracted, and Western blotting using anti-cyAbrB1 antibody was performed. Different amounts of total proteins from C100 cultured in the absence of aTc were loaded to show the linearity of the results. (C) Time course analysis of CalA/cyAbrB1 knockdown. Each strain was cultivated for the indicated times in the presence of 50 ng/ml aTc. Subsequently, total protein was extracted, and Western blotting using anti-cyAbrB1 antibody was performed.

FIG 2

Upregulation of some genes related to heterocyst differentiation following knockdown of calA/cyabrB1. After 48 h of cultivation in the absence or presence of 50 ng/ml aTc, RNA was extracted, and RT-qPCR was performed. rnpB was used for normalization. Data represent means ± standard deviations (SD) (n = 3 from independent cultures). Amounts of each gene relative to those in C100 cells grown in nitrate-containing medium bubbled with air containing 5% (vol/vol) CO₂ in the absence of the inducer are shown. (A) Each strain was grown in nitrate-containing medium and bubbled with air containing 5% (vol/vol) CO₂. (B) Strain C104 was grown in nitrate-containing medium bubbled with air containing 1% (vol/vol) CO₂ or in ammonium-containing medium bubbled with air containing 5% (vol/vol) CO₂.

FIG 3

Knockdown of calA/cyabrB1 in the hetR mutant background. Cells were cultured in nitrate-containing medium bubbled with air containing 5% (vol/vol) CO₂ in the absence or presence of aTc for 48 h. (A) Effect of hetR inactivation on expression of calA/cyabrB1 and genes related to heterocyst formation. RNA was extracted, and RT-qPCR was performed. rnpB was used for normalization. Data represent means ± SD (n = 3 from independent cultures). Amounts of each gene relative to those in C100 cells cultured in nitrate-containing medium bubbled with air containing 5% (vol/vol) CO₂ in the absence of the inducer are shown. (B) Confirmation of CalA/cyAbrB1 knockdown. Total protein was extracted, and Western blotting using anti-cyAbrB1 antibody was performed. (C) No heterocyst formation by calA/cyabrB1 knockdown in a hetR-deficient background. Cells of C104h were microphotographed.

FIG 4

Specific and redundant functions of cyabrB genes. Cells were cultured in nitrate-containing medium bubbled with air containing 1% (vol/vol) CO₂ in the absence or presence of aTc for 48 h. (A) Formation of heterocysts by calA/cyabrB1 knockdown and calB/cyabrB2 knockout. Cells of C104B2 or DR2080 were microphotographed. (B) RNA was extracted, and RT-qPCR was performed. rnpB was used for normalization. Data represent the means ± SD (n = 3 from independent cultures). Amounts of each gene relative to those in C100 cells grown in nitrate-containing medium bubbled with air containing 5% (vol/vol) CO₂ in the absence of the inducer are shown.

FIG 5

Direct binding of CalA/cyAbrB1 to promoters of hetP and hepA. (A) Purification of His-CalA/cyAbrB1. Purified His-CalA/cyAbrB1 was subjected to 15% SDS-PAGE. Lane M, protein molecular weight marker; lane His-CalA/cyAbrB1, purified His-CalA/cyAbrB1. (B) Electrophoretic mobility shift assay with His-CalA/cyAbrB1. His-CalA/cyAbrB1 was mixed with 3 nM DNA probe (promoter region of hetP). Nonlabeled DNAs (PhetP, PhepA, or the internal region of calA/cyabrB1 [cyabrB1RT]) were added.

FIG 6

Effect of calA/cyabrB1 overexpression on heterocyst development. Cells of the negative-control strain C100 and the calA/cyabrB1 overexpression strain T121 were cultured in the absence or presence of 200 ng/ml aTc. (A) Impaired growth under nitrogen-depleted conditions by calA/cyabrB1 overexpression. Cells were cultured in nitrogen-free or nitrate-containing medium, and the OD₇₅₀ was monitored. Data represent means ± SD (n = 3 from independent cultures). (B) Effect of calA/cyabrB1 overexpression on heterocyst formation. Cells of C100 and T121 cultured for 24 h in the absence or presence of the inducer were microphotographed. (C) Effect of calA/cyabrB1 overexpression on expression of genes related to heterocyst formation. After 8 h of nitrogen depletion, RNA was extracted, and RT-qPCR was performed. rnpB was used for normalization. Data represent means ± SD (n = 3 from independent culture). Amounts of each gene relative to those in C100 cells grown in nitrate-free medium in the absence of the inducer are shown. Overexpression of CalA/cyAbrB1 was confirmed by Western blotting.