Functional Overlap of <i>hetP</i> and <i>hetZ</i> in Regulation of Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

Abstract

HetR plays a key role in regulation of heterocyst differentiation and patterning in <i>Anabaena</i> It directly regulates genes involved in heterocyst differentiation (such as <i>hetP</i> and <i>hetZ</i>), genes involved in pattern formation (<i>patA</i>), and many others. In this study, we investigated the functional relationship of <i>hetP</i> and <i>hetZ</i> and their role in HetR-dependent cell differentiation. Coexpression of <i>hetP</i> and <i>hetZ</i> from the promoter of <i>ntcA</i>, which encodes the global nitrogen regulator, enabled a <i>hetR</i> mutant to form heterocysts with low aerobic nitrogenase activity. Overexpression of <i>hetZ</i> restored heterocyst differentiation in a <i>hetP</i> mutant and vice versa. Overexpression of <i>hetR</i> restored heterocyst formation in either a <i>hetP</i> or a <i>hetZ</i> mutant but not in a <i>hetZ hetP</i> double mutant. The functional overlap of <i>hetP</i> and <i>hetZ</i> was further confirmed by reverse transcription-quantitative PCR (RT-qPCR) and transcriptomic analyses of their effects on gene expression. In addition, yeast two-hybrid and pulldown assays showed the interaction of HetZ with HetR. HetP and HetZ are proposed as the two major factors that control heterocyst formation in response to upregulation of <i>hetR</i><b>IMPORTANCE</b> Heterocyst-forming cyanobacteria contribute significantly to N₂ fixation in marine, freshwater, and terrestrial ecosystems. Formation of heterocysts enables this group of cyanobacteria to fix N₂ efficiently under aerobic conditions. HetR, HetP, and HetZ are among the most important factors involved in heterocyst differentiation. We present evidence for the functional overlap of <i>hetP</i> and <i>hetZ</i> and for the key role of the HetR-HetP/HetZ circuit in regulation of heterocyst differentiation. The regulatory mechanism based on HetR, HetP, and HetZ is probably conserved in all heterocyst-forming cyanobacteria.