Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

Abstract

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petEhetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.

Figure 1

Identification of four potential transcription start sites of the hetR gene by primer extension. RNA isolated from cells before and 18 h after nitrogen starvation was used with specific labeled primers.

Figure 2

Northern gel analysis of the early expression of hetR after nitrogen starvation in wild type and the hetR point mutant 216. The lane labels refer to hours postinduction. The 0.5-h 216 lane is blank, likely to be a result of sample degradation, and the 1.9-kb band in the 216 3.0- and 4.0-h lanes is distorted because of an air bubble during transfer of the RNA.

Figure 3

Structure of the plasmid pPetHetR, containing the Anabaena hetR gene, whose expression is driven by the petE promoter. Note that a cat gene reporter is located downstream of the hetR gene.

Figure 4

Dependence of CAT antigen induction as a function of added copper ion in wild-type Anabaena 7120 containing pPetHetR. Cells were grown for 24 h in BG-11 medium containing 1 mM (NH₄)₂S0₄, and CAT antigen was assayed as described in Materials and Methods.

Figure 5

Chlorophyll fluorescence in Anabaena cultures 2 days after the addition of 2 μM Cu²⁺ to induce expression of the hetR gene. In each case, the culture contained wild-type Anabaena carrying the plasmid pPetHetR, and the fluorescence was viewed with a wide bandpass filter. (A) The culture contained 1 mM (NH₄)₂S0₄. (B) The culture contained 17 mM nitrate. (C) The culture contained no fixed nitrogen. The heterocysts can be identified by the low fluorescence attributable to the loss of chlorophyll from photosystem II and the degradation of the phycobiliproteins.

Figure 6

Model of the relationships among some of the gene products that control heterocyst differentiation in Anabaena. Normally, the environmental signal of low fixed nitrogen is perceived by the NtcA protein, as modified by the regulator P_II. NtcA activates transcription of many genes, eventually including hetR. When modified by PatA, HetR activates expression of itself and other genes leading to heterocyst differentiation. HetR action is countered by both PatS and HetN.