patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

Abstract

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.

FIG. 1.

Diagram of patS minigene constructs. Each PCR product results in a promoter upstream region (open arrows) fused at the start codon to a patS minigene sequence, ending with a UAG stop codon encoded by the 5′ end of the reverse primer. The 3′ ends of the reverse primers are complementary to the promoter template. (A) Construction of P_petE-patS4 to P_petE-patS8. pAM2269 was used as the PCR template for P_petE. The forward primer was AMO-388 for all five constructions. The PatS peptides encoded by the reverse primers are shown. (B) To generate P_patS-patS5, pAM1951 was used as the PCR template for P_patS and the patS5 sequence was encoded on the reverse primer AMO-432. AMO-367 was the forward primer. (C) To generate P_rbcL-patS5, pAM1954 was used as the PCR template for P_rbcL and the patS5 sequence was encoded on the reverse primer AMO-421. AMO-367 was the forward primer. (D) To generate P_hepA-patS5, pAM1630 was used as the PCR template for P_hepA and the patS5 sequence was encoded on the reverse primer AMO-557. GTL-T7 was the forward primer. The closed arrows indicate PCR primers. *, stop codon. Diagrams are not to scale.

FIG. 2.

PatS-5 does not function as a cell-to-cell signal. Plasmids containing patS17 and patS5 expressed from the proheterocyst-specific promoter P_hepA were used to complement the patS deletion strain AMC451. The heterocyst pattern was determined 24 h after nitrogen step-down for the wild type (A), strain AMC451 (B), AMC451 containing pAM1715 (P_hepA-patS17) (C), and AMC451 containing pAM2816 (P_hepA-patS5-gfp) (D).