Ancient association of cyanobacterial multicellularity with the regulator HetR and an RGSGR pentapeptide‐containing protein (PatX)

Abstract

One simple model to explain biological pattern postulates the existence of a stationary regulator of differentiation that positively affects its own expression, coupled with a diffusible suppressor of differentiation that inhibits the regulator's expression. The first has been identified in the filamentous, heterocyst-forming cyanobacterium, Anabaena PCC 7120 as the transcriptional regulator, HetR and the second as the small protein, PatS, which contains a critical RGSGR motif that binds to HetR. HetR is present in almost all filamentous cyanobacteria, but only a subset of heterocyst-forming strains carry proteins similar to PatS. We identified a third protein, PatX that also carries the RGSGR motif and is coextensive with HetR. Amino acid sequences of PatX contain two conserved regions: the RGSGR motif and a hydrophobic N-terminus. Within 69 nt upstream from all instances of the gene is a DIF1 motif correlated in Anabaena with promoter induction in developing heterocysts, preceded in heterocyst-forming strains by an apparent NtcA-binding site, associated with regulation by nitrogen-status. Consistent with a role in the simple model, PatX is expressed dependent on HetR and acts to inhibit differentiation. The acquisition of the PatX/HetR pair preceded the appearance of both PatS and heterocysts, dating back to the beginnings of multicellularity.

Elements of the heterocyst‐specific transcriptome unravelled by co‐expression analysis in Nostoc sp. PCC 7120

“…S6). Consistent with recent observations (Elhai and Khudyakov, ; Wegelius et al ., ), a conserved G (likely associated to the −10 box of these promoters) is located 16–18 nt downstream of the DIF1 motif (Supporting Information Fig. S5 and S6).…”

“…Consistent with its role in early inhibition of differentiation, patS appeared in the E‐DIF cluster. Recently, another gene encoding a RGTGR‐containing peptide that would act as a second, PatS‐like inhibitor of heterocyst differentiation, has been described and named as patX (Elhai and Khudyakov, ). According to our data, asl2332 , the gene encoding PatX, appears also within the E‐DIF cluster, suggesting that the, perhaps redundant, inhibitory effects of both PatS and PatX would take place in early stages of differentiation.…”

“…Recently, it has been demonstrated that patS and hetN are not functionally equivalent when their promoter regions are switched, therefore suggesting their respective timing of expression is critical for proper function (Rivers et al, 2018). Two other genes described as participating in patterning of heterocysts, patC (Corrales-Guerrero et al, 2014) and patX (Elhai and Khudyakov, 2018), also appeared in the E-DIF group. Therefore, according to the transcriptional profiles described here, up to three elements would act as negative regulators during initial steps of differentiation in Nostoc sp.…”

Expression from DIF1-motif promoters of hetR and patS is dependent on HetZ and modulated by PatU3 during heterocyst differentiation

“…Heterocyst differentiation and pattern formation largely depend on the key regulator HetR [11] and RGSGR-containing peptides, which are derived from PatS [12, 13], PatX [14] or HetN [15], representing an example of the most ancient activator-inhibitor (reaction-diffusion) patterning process [16–18]. In Anabaena 7120, PatS is the main source of morphogen for de novo pattern formation [13], while HetN is required for maintenance of the pattern [19].…”

“…Before the consensus HetR-recognition sequence was identified, DIF + (later called DIF1) motif (TCCGGA) had been bioinformatically identified in sequences upstream of hetR and several other genes in Anabaena 7120 [33]. More recently, the DIF1 motif was proposed as a consensus regulatory sequence (centered at −35 region) for patS and patX in heterocyst-forming cyanobacteria [34]. The role of DIF1 motif in expression of the nsiR promoter [33] and a synthetic minimal promoter has been reported [35].…”