Manipulation of Pattern of Cell Differentiation in a hetR Mutant of Anabaena sp. PCC 7120 by Overexpressing hetZ Alone or with hetP

Abstract

In the filamentous cyanobacterium, Anabaena sp. PCC 7120, single heterocysts differentiate at semi-regular intervals in response to nitrogen stepdown. HetR is a principal regulator of heterocyst differentiation, and hetP and hetZ are two genes that are regulated directly by HetR. In a hetR mutant generated from the IHB (Institute of Hydrobiology) substrain of PCC 7120, heterocyst formation can be restored by moderate expression of hetZ and hetP. The resulting heterocysts are located at terminal positions. We used a tandem promoter, PrbcLPpetE, to express hetZ and hetP strongly in the hetR mutant. Co-expression of hetZ and hetP enabled the hetR mutant to form multiple contiguous heterocysts at both terminal and intercalary positions. Expression of hetZ, alone resulted in terminally located heterocysts, whereas expression of hetP, alone produced enlarged cells in strings. In the absence of HetR, formation of heterocysts was insensitive to the peptide inhibitor, RGSGR.

Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120

“…Similar instability has been seen with strains overexpressing HetR [62] or HetZ and HetP [63] and strains with mutated hetN [25]. In our experiments, we tried to avoid this problem by using conditional mutants and maintaining them in a medium that blocked heterocyst development completely and thus presumably eliminated negative selection pressure on mutants overproducing heterocysts [43,63]. Nevertheless, a high mutation rate was still observed, resulting in the rapid accumulation of Het − and PatA-like mutants producing only terminal heterocysts (Figure 3).…”

“…The developmental chaos that results from patX patS and hetN patS double mutants produces strong selective pressure for the rapid accumulation of suppressor mutations to escape from lethal terminal differentiation. Similar instability has been seen with strains overexpressing HetR [62] or HetZ and HetP [63] and strains with mutated hetN [25]. In our experiments, we tried to avoid this problem by using conditional mutants and maintaining them in a medium that blocked heterocyst development completely and thus presumably eliminated negative selection pressure on mutants overproducing heterocysts [43,63].…”

Preventing Accidental Heterocyst Development in Cyanobacteria

“…Indeed, these two genes, as direct targets of HetR, can bypass the requirement of the latter for heterocyst differentiation. When overexpressed alone or together, they can partly or completely rescue heterocyst differentiation in the absence of hetR (14)(15)(16)(17)(18). One hypothesis would be that heterocysts that developed under calA overexpression progressed beyond the steps controlled by hetZ and hetP but were blocked at the morphogenesis step through the action of CalA on hepA (Fig.…”

“…How HetP and HetZ regulate heterocyst development remains unknown, but they have redundant functions, since depending on the expression levels, they can bypass, alone or together, the requirement of HetR in heterocyst development. HetP and likely HetZ represent a transition step from initiation toward commitment during heterocyst development (15)(16)(17)(18) (Fig. 1).…”

Developmental Biology in Cyanobacteria

“…Heterocyst pattern formation or maintenance is addressed in two research articles and one review. Zhang and Xu inquire about the role of some regulatory genes (hetR, hetZ, hetP) in heterocyst differentiation [3], and Fukushima and Ehira describe a novel Ser/Thr kinase necessary to keep a normal heterocyst pattern in the diazotrophic filament [4]. Additionally, Arbel-Goren et al review a stochastic Turing model that offers a robust description of heterocyst pattern formation [5].…”