A Tripartite, Hierarchical Sigma Factor Cascade Promotes Hormogonium Development in the Filamentous Cyanobacterium Nostoc punctiforme

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.

Identification of a hormogonium polysaccharide‐specific gene set conserved in filamentous cyanobacteria

“…Because transcription of sigC is dependent on sigJ (Gonzalez et al, 2019), this result indicates that transcription of hpsQ is likely sigCdependent. The hpsR gene encodes a putative glycosyl transferase F I G U R E 1 Genomic context and expression of hps loci (a) Gene maps of the hps loci with corresponding RNAseq based read coverage data, derived from (Gonzalez et al, 2019), at 0 and 12 hr (as indicated) post hormogonium induction. ± indicates reads mapping to the sense and antisense strands of the chromosome, respectively, with the number range in brackets at the upper left for each locus indicating the read coverage scale.…”

“…Black triangles indicate the site of transposon insertions. (b) Heat map depicting the RNAseq based expression of hps genes, derived from (Gonzalez et al, 2019), over a five point time course of hormogonium development in both the wild type and each hormogonium-specific sigma factor deletion strain. Expression = Log2(average normalized expression for each strain and time point/average normalized expression of the wild type at 0 hr) and is immediately downstream and in the same orientation as Npun_R1507, which codes for a putative carboxy-terminal protease ( Figure 1A).…”

“…Expression = Log2(average normalized expression for each strain and time point/average normalized expression of the wild type at 0 hr) and is immediately downstream and in the same orientation as Npun_R1507, which codes for a putative carboxy-terminal protease ( Figure 1A). RNAseq read coverage indicates that both genes may be co-transcribed ( Figure 1A), however, transcription of Npun_R1507 is largely static during hormogonium development (Gonzalez et al, 2019), whereas hpsR is upregulated in a sigJ-dependent manner ( Figure 1B). A consensus J-box (GGGAATACT) (Gonzalez et al, 2019) is located 33 bp upstream of hpsR ( Figure 1A), providing further evidence that hpsR is transcribed as a sigJ-dependent, monocistronic transcript in developing hormogonia.…”

“…RNAseq read coverage indicates that both genes may be co-transcribed ( Figure 1A), however, transcription of Npun_R1507 is largely static during hormogonium development (Gonzalez et al, 2019), whereas hpsR is upregulated in a sigJ-dependent manner ( Figure 1B). A consensus J-box (GGGAATACT) (Gonzalez et al, 2019) is located 33 bp upstream of hpsR ( Figure 1A), providing further evidence that hpsR is transcribed as a sigJ-dependent, monocistronic transcript in developing hormogonia. The hpsS-U locus contains two genes encoding putative glycosyl transferases, hpsS and hpsT, and a third gene, hpsU, that codes for a homolog of E. coli WcaF, an acetyltransferase involved in the synthesis of the extracellular polysaccharide colanic acid (Stevenson et al, 1996).…”

Identification and characterization of novel filament-forming proteins in cyanobacteria

“…PCC 7942 (hereafter Synechococcus) MreB is essential, where partially segregated mutants display a coccoid morphology resembling the morphology of E. coli mreB deletion strains 16,17 . In Nostoc punctiforme ATCC 29113, the MreBCD operon was shown to be regulated by the hormogonium-specific sigma factor SigJ and is likely involved in the transition of coccoid vegetative cells to the more rod-shaped cells that are characteristic to hormogonia 18 .…”