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ABSTRACT Filamentous, heterocyst-forming cyanobacteria belonging to taxonomic subsections IV and V are developmentally complex multicellular organisms capable of differentiating an array of cell and filament types, including motile hormogonia. Hormogonia exhibit gliding motility that facilitates dispersal, phototaxis, and the establishment of nitrogen-fixing symbioses. The gene regulatory network (GRN) governing hormogonium development involves a hierarchical sigma factor cascade, but the factors governing the activation of this cascade are currently undefined. Here, using a forward genetic approach, we identified hrmK , a gene encoding a putative hybrid histidine kinase that functions upstream of the sigma factor cascade. The deletion of hrmK produced nonmotile filaments that failed to display hormogonium morphology or accumulate hormogonium-specific proteins or polysaccharide. Targeted transcriptional analyses using reverse transcription-quantitative PCR (RT-qPCR) demonstrated that hormogonium-specific genes both within and outside the sigma factor cascade are drastically downregulated in the absence of hrmK and that hrmK may be subject to indirect, positive autoregulation via sigJ and sigC . Orthologs of HrmK are ubiquitous among, and exclusive to, heterocyst-forming cyanobacteria. Collectively, these results indicate that hrmK functions upstream of the sigma factor cascade to initiate hormogonium development, likely by modulating the phosphorylation state of an unknown protein that may serve as the master regulator of hormogonium development in heterocyst-forming cyanobacteria. IMPORTANCE Filamentous cyanobacteria are morphologically complex, with several representative species amenable to routine genetic manipulation, making them excellent model organisms for the study of development. Furthermore, two of the developmental alternatives, nitrogen-fixing heterocysts and motile hormogonia, are essential to establish nitrogen-fixing symbioses with plant partners. These symbioses are integral to global nitrogen cycles and could be artificially recreated with crop plants to serve as biofertilizers, but to achieve this goal, detailed understanding and manipulation of the hormogonium and heterocyst gene regulatory networks may be necessary. Here, using the model organism Nostoc punctiforme , we identify a previously uncharacterized hybrid histidine kinase that is confined to heterocyst-forming cyanobacteria as the earliest known participant in hormogonium development. 

ABSTRACT Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and frequently, nitrogen fixation as well. As a result, they contribute substantially to global primary production and nitrogen cycles. Furthermore, the multicellular filamentous cyanobacteria in taxonomic subsections IV and V are developmentally complex, exhibiting an array of differentiated cell types and filaments, including motile hormogonia, making them valuable model organisms for studying development. To investigate the role of sigma factors in the gene regulatory network (GRN) controlling hormogonium development, a combination of genetic, immunological, and time-resolved transcriptomic analyses were conducted in the model filamentous cyanobacterium Nostoc punctiforme , which, unlike other common model cyanobacteria, retains the developmental complexity of field isolates. The results support a model where the hormogonium GRN is driven by a hierarchal sigma factor cascade, with sigJ activating the expression of both sigC and sigF, as well as a substantial portion of additional hormogonium-specific genes, including those driving changes to cellular architecture. In turn, sigC regulates smaller subsets of genes for several processes, plays a dominant role in promoting reductive cell division, and may also both positively and negatively regulate sigJ to reinforce the developmental program and coordinate the timing of gene expression, respectively. In contrast, the sigF regulon is extremely limited. Among genes with characterized roles in hormogonium development, only pilA shows stringent sigF dependence. For sigJ -dependent genes, a putative consensus promoter was also identified, consisting primarily of a highly conserved extended −10 region, here designated a J-Box, which is widely distributed among diverse members of the cyanobacterial lineage. IMPORTANCE Cyanobacteria are integral to global carbon and nitrogen cycles, and their metabolic capacity coupled with their ease of genetic manipulation make them attractive platforms for applications such as biomaterial and biofertilizer production. Achieving these goals will likely require a detailed understanding and precise rewiring of these organisms’ GRNs. The complex phenotypic plasticity of filamentous cyanobacteria has also made them valuable models of prokaryotic development. However, current research has been limited by focusing primarily on a handful of model strains which fail to reflect the phenotypes of field counterparts, potentially limiting biotechnological advances and a more comprehensive understanding of developmental complexity. Here, using Nostoc punctiforme , a model filamentous cyanobacterium that retains the developmental range of wild isolates, we define previously unknown definitive roles for a trio of sigma factors during hormogonium development. These findings substantially advance our understanding of cyanobacterial development and gene regulation and could be leveraged for future applications.