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.

Molecular circuit of heterocyst differentiation in cyanobacteria

“…cyABrB1 (calA) is one such regulator that inhibits the heterocyst formation. It is observed that the conditional knockdown of cyABrB1, using CRISPR interference, leads to the formation of heterocyst, even in the presence of nitrate [8]. It is found that the knockdown of cyABrB1 leads to the upregulation of hetP and hepA (promoters of heterocyst differentiation), which are otherwise induced by HetR.…”

“…It is found that the knockdown of cyABrB1 leads to the upregulation of hetP and hepA (promoters of heterocyst differentiation), which are otherwise induced by HetR. It is proposed that the ratio of HetR and cyABrB1 might be playing an important role in the commitment of vegetative cells toward heterocyst formation [8]. Likewise, PknE kinase (alr3732) is also known to inhibit HetR during a later stage in differentiation [9].…”

Preventing Accidental Heterocyst Development in Cyanobacteria

“…Higo et al used the CRISPRi approach to switch the expression of calA to a level low enough to obtain some phenotypes (3). Unfortunately, the authors could not confirm the essential function of calA, as one would expect, albeit the growth of the corresponding mutant under interference conditions was significantly inhibited.…”

“…At least two direct targets of CalA are identified, hetP and hepA, as CalA can bind to their promoter regions (3). The expression of these two genes is increased once calA expression is turned down, and this regulation is independent of HetR.…”

“…Strangely, while hepA expression is negatively affected by calA overexpression, that of hetP is little changed. This result is puzzling since both hetP and hepA are reported to be under the dual control of HetR and CalA (3,28). The authors argue that HetR binds more strongly to the hetP promoter than that of hepA and thus outcompetes CalA for binding to the promoter region of hetP (3).…”