Interaction network among factors involved in heterocyst-patterning in cyanobacteria

Abstract

The genetically regulated pattern of heterocyst formation in multicellular cyanobacteria represents the simplest model to address how patterns emerge and are established, the signals that control them, and the regulatory pathways that act downstream. Although numerous factors involved in this process have been identified, the mechanisms of action of many of them remain largely unknown. The aim of this study was to identify specific relationships between 14 factors required for cell differentiation and pattern formation by exploring their putative physical interactions in the cyanobacterium model Nostoc sp. PCC 7120 and by probing their evolutionary conservation and distribution across the cyanobacterial phylum. A bacterial two-hybrid assay indicated that 10 of the 14 factors studied here are engaged in more than one protein-protein interaction. The transcriptional regulator PatB was central in this network as it showed the highest number of binary interactions. A phylum-wide genomic survey of the distribution of these factors in cyanobacteria showed that they are all highly conserved in the genomes of heterocyst-forming strains, with the PatN protein being almost restricted to this clade. Interestingly, eight of the factors that were shown to be capable of protein interactions were identified as key elements in the evolutionary genomics analysis. These data suggest that a network of 12 proteins may play a crucial role in heterocyst development and patterning. Unravelling the physical and functional interactions between these factors during heterocyst development will certainly shed light on the mechanisms underlying pattern establishment in cyanobacteria. Response to Reviewers:Dear editor, in this revised version of our manuscript we introduced all the corrections asked by the editor and Reviewer 1. The modifications in the text are marked in yellow, We thank the reviewers and the communicating editor for their valuable reading of our publication. We hope that this revised manuscript will be suited for publication.

Evidence that the PatB (CnfR) factor acts as a direct transcriptional regulator to control heterocyst development and function in the cyanobacterium Nostoc PCC 7120

“…His 6 ‐tagged PatB was purified as described in (Xu et al, 2022), with the following modifications. BL21DE3 cells transformed with pLIC07‐PatB were used for protein production and grown 6 h after induction performed with 1 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) addition.…”

“…Our data rather support the hypothesis that the absence of HTH domain could be rescued in the heterocyst. We have previously demonstrated that PatB is able to establish physical interactions with several other factors involved in heterocyst formation (HetR, HetL, HetF', HetP, and PatL) (Xu et al, 2022). It would be tempting to think that one of these interactions could help the PatB ΔHTH variant to regulate F I G U R E 3 Microscope images of the patB deletion strain harboring the reporter fusion P nifB ::gfp and the patB gene or its variants expressed under the PpetE promoter.…”

“…PCC 7502, Crocosphaera watsonii WH 8501, Cyanothece sp. PCC 7425, and Gloeothece verrucosa PCC 7822) (Xu et al, 2022). Nevertheless, its function in these strains is so for unknown.…”

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…The bacterial two-hybrid system (BACTH), a genetic approach to protein-protein interaction (PPI) that is based on the reconstitution in E. coli of the adenylate cyclase of Bordetella pertussis from its T18 and T25 domains, fused to the proteins of interest [41,42], has been successfully used to identify components and unravel the molecular details of interaction networks involved in cell division or heterocyst patterning in cyanobacteria [43][44][45][46]. We recently used the BACTH system to prove interactions between PipX and the ribosomeassembly GTPase EngA, two proteins whose interactions in cyanobacteria were previously inferred by synteny [47] and in vivo [48] approaches.…”

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…The bacterial two-hybrid system (BACTH), a genetic approach to protein-protein interaction (PPI) based on the reconstitution in E. coli of the adenylate cyclase of Bordetella pertussis from its T18 and T25 domains fused to the proteins of interest [42] has been successfully used to identify components and unravel molecular details of interactions networks involved in cell division or heterocyst patterning in cyanobacteria [43][44][45][46]. We recently used the BACTH system to prove interactions between PipX and the ribosome-assembly GTPase EngA, two proteins whose interactions in cyanobacteria were previously inferred by synteny and in vivo approaches [47,48].…”