The Hybrid Histidine Kinase HrmK Is an Early-Acting Factor in the Hormogonium Gene Regulatory Network

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.

Keywords: Nostoc punctiforme; cell motility; cyanobacteria; development; developmental biology; gliding motility; histidine kinase; hormogonia; two-component regulatory systems.

FIG 1

Characterization of hrmK. (A) Gene map of the hrmK locus. Triangles indicate the sites of transposon insertions. HisKA, His kinase A (phosphoacceptor) domain; HATPase, ATPase domains of histidine kinase; REC, response regulator receiver domain. (B) Plate motility assays of the wild-type strain, ΔhrmK mutant, and ΔhrmK mutant with hrmK expressed in trans from a shuttle vector (+hrmK) (as indicated). Images were taken at 2 days and 7 days postinduction. (C) Light micrographs of the filament morphology for the wild-type and ΔhrmK mutant strains (as indicated) at 0 h and 24 h post-hormogonium induction (as indicated). Carets indicate the presence of heterocysts attached to filaments. (D) RT-qPCR of hrmK in the wild-type, ΔsigJ mutant, and ΔsigC mutant strains 0 and 18 h after induction for hormogonia. *, P < 0.05; **, P < 0.01, as determined by two-tailed Student’s t test between the wild type and each deletion strain at the corresponding time point. !, P < 0.05; as determined by two-tailed Student’s t test between 0 and 18 h for the same strain.

FIG 2

Effect of hrmK on accumulation of HmpD, PilA, and HPS. (A) Immunoblot analysis of HmpD, PilA, and RbcL in the wild-type and ΔhrmK mutant strains (as indicated) 0 and 24 h after hormogonium induction. RbcL is the large subunit of RuBisCO and serves as a protein loading control. (B) Fluorescent lectin staining of HPS in the wild-type and ΔhrmK mutant strains (as indicated). Depicted are merged images of fluorescence micrographs acquired using a 10× lens objective from cellular autofluorescence (red) and HPS (yellow) (strains as indicated) 0 and 24 h after hormogonium induction.

FIG 3

RT-qPCR of hormogonium-specific gene expression (as indicated) in the wild-type and ΔhrmK mutant strains 0 and 18 h after induction for hormogonia. *, P < 0.05; **, P < 0.01, as determined by two-tailed Student’s t test between the wild-type and ΔhrmK mutant strains at the corresponding time point. !, P < 0.05; !!, P < 0.01, as determined by two-tailed Student’s t test between 0 and 18 h for the same strain.

FIG 4

Evolutionary conservation of HrmK in cyanobacteria. Shown is a heat map depicting the percent identity for orthologs of N. punctiforme HrmK in cyanobacteria, derived from data reported by Cho et al. (26). The species organization and phylogenetic tree are based on the phylogeny reported by Shih et al. (36) but depicting the finding, as reported by Schirrmeister et al. (37), that most extant cyanobacteria are derived from a filamentous ancestor. For the phylogenetic tree, green indicates filamentous and black indicates unicellular.

FIG 5

A model depicting the major findings of this report on the role of hrmK in the hormogonium GRN. Arrows indicate positive regulation, and lines with bars indicate negative regulation. The thickness of the arrows represents a combination of the number of genes regulated and the stringency of the regulation.