Epistasis analysis of four genes from Anabaena sp. strain PCC 7120 suggests a connection between PatA and PatS in heterocyst pattern formation

Abstract

The hetR, patA, hetN, and patS genes are part of a regulatory network that regulates the differentiation and patterning of heterocysts in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. In this report, the epistatic interactions of mutant alleles of these four genes have been used to refine our understanding of their relationships to one another. The hetR gene was necessary for differentiation in genetic backgrounds that normally give rise to excessive differentiation, supporting its role as the master regulator of differentiation and indicating that HetR directly regulates factors in addition to hetR and patS genes that regulate differentiation. A functional patS gene was necessary for the delayed multiple-contiguous-heterocyst phenotype observed in hetN mutants as well as for the relative lack of intercalary heterocysts in patA mutants. Epistasis results with mutant alleles of these three genes suggested that PatA attenuates the negative effects of both PatS and HetN on differentiation and promotes differentiation independent of its antagonistic effects on PatS and HetN activity. Cooverxpression of patS and hetR in a synthetic operon indicated that patS acts at a point downstream of hetR transcription in the regulatory network controlling differentiation. A model for the regulation of differentiation that is consistent with these and previous findings is presented.

FIG. 1.

Phenotypes of single mutant strains 48 h postinduction. PCC 7120 (A), hetR mutant strain UHM103 (B), patA mutant strain UHM101 (C), patS mutant strain UHM114 (D), hetN mutant strain 7120PN (E).

FIG. 2.

Mch termini of a hetN patA double mutant strain. UHM112 48 h postinduction in copper-free medium (A). Comparison of the percentages of filament termini from strains UHM101 and UHM112, which have the indicated numbers of contiguous heterocysts at different times postinduction (B).

FIG. 3.

Vegetative-cell intervals versus time postinduction and the phenotype of a patS hetN patA mutant. Average numbers of vegetative cells between clusters of heterocysts (average vegetative-cell interval) at various times postinduction (A). PCC 7120 (⧫), UHM114 (▪), UHM113 (▴), UHM101 sheared to an average filament length of 10 cells prior to induction (✖), UHM108 (✚), UHM100 (•). UHM108 48 h postinduction in copper-free medium (B). UHM100 48 h postinduction in copper-free medium (C). UHM108 in copper-free medium containing nitrate (D).

FIG. 4.

Heterocyst formation by a patS patA double mutant. UHM113 in medium containing nitrate (A). UHM113 24 h postinduction (B).

FIG. 5.

Model depicting the genetic interactions of genes in this study and their roles in cell fate determination. See the text for details.