ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

Abstract

In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS-and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS-and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetCindependent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. IMPORTANCEHow chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was shown to be required for signaling between heterocysts, which supply fixed nitrogen to the organism, and other cells, as well as for conferring immunity to self-signaling on developing heterocysts. Differentiation of heterocysts by filamentous cyanobacteria represents a simple but elegant model of biological patterning. In response to a shortage of combined nitrogen, linear filaments of Anabaena sp. strain PCC 7120 (here, Anabaena) form a periodic pattern of single, terminally differentiated, nitrogen-fixing heterocysts separated by approximately 10 totipotent vegetative cells. The spacing between heterocysts reflects the metabolic interdependence of the two cell types; fixed nitrogen is supplied to vegetative cells from heterocysts, and in return, heterocysts receive a source of carbon and reductant to compensate for their lack of photosystem II and the Calvin cycle (for a review of heterocyst differentiation, see reference 1). As filaments lengthen by growth and division of vegetative cells, subsequent rounds of heterocyst differentiation place new hetero...

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

“…Epistasis analysis similar to that done here for sepJ and patS or hetN has been recently performed for hetC and patS or hetN . The results of these analyses are consistent with the idea that the HetC ABC exporter participates in export from proheterocysts of the PatS morphogen (Corrales‐Guerrero et al ., ) or of the PatS morphogen and a HetN‐dependent signal (Videau et al ., ). The existence of a transporter specific for export of the regulators from the (pro)heterocysts to their neighboring vegetative cells could make transfer not limited to a SepJ‐dependent pathway.…”

“…Based on expression of a P hetR ‐ gfp transcriptional fusion in a sepJ ( fraG ) background, it has been suggested that SepJ is not needed for proper heterocyst pattern formation (Nayar et al ., ). However, the results described in this work and those of other recent studies (Corrales‐Guerrero et al ., 2014b; 2015; Videau et al ., ) have unraveled possible relations between SepJ and PatS and HetN, as well as between HetC and PatS and HetN, affecting heterocyst pattern formation. Heterocyst differentiation aborts early in sepJ (Flores et al ., ; Nayar et al ., ) and hetC (Khudyakov and Wolk, ; Xu and Wolk, ) inactivation mutants, an effect that could result from accumulation of inhibitory PatS that is not exported from proheterocysts.…”

“…Material reacting with antibodies raised against PatS‐5 has been detected in cells proximal to patS ‐producing proheterocysts, but not in the proheterocysts, consistent with the intercellular transfer of a PatS‐derived morphogen (Corrales‐Guerrero et al ., ). The mechanism of transfer is however unknown, although recent data has suggested the involvement of the ABC transporter HetC in export of PatS from the heterocysts (Corrales‐Guerrero et al ., ; Videau et al ., ).…”

The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120

“…Media for Anabaena growth on ammonia as the nitrogen source was prepared as previously described (47). Heterocyst percentages were determined as previously described, and all results are expressed as the average of three replicates ± SD (48). The vegetative cell interval between heterocysts was determined by counting 300 intervals as previously described (47).…”

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

“…Transposon mutagenesis, screening for mutants unable to grow diazotrophically, and determination of the transposon insertion site were performed as previously described (31,32). The ability of strains to grow diazotrophically was assessed visually following 2 weeks of growth on BG-11 medium lacking a combined nitrogen source as previously described (33). Expression from the copper-inducible petE promoter was achieved with the addition of copper to a final concentration of 2 M (34).…”