Competition and interdependence define multifaceted interactions of symbiotic Nostoc sp. and Agrobacterium sp. under inorganic carbon limitation

Cyanobacteria of the Nostoc genus are capable of forming symbiotic relationships with plants, thus transitioning to a heterotrophic lifestyle in return for providing bioavailable nitrogen to the host. The diazotrophic photoautotrophs also serve as a hub for specialized heterotrophic bacterial communities whose physiological contributions are poorly understood. By comparing the axenic strain Nostoc punctiforme PCC 73102 and the related strains Nostoc sp. KVJ2 and KVJ3, which still maintain their heterotrophic microbiome, we were able to demonstrate an almost obligate dependence of the cyanobacteria on the heterotrophic partners under carbon-limiting conditions. Detailed analysis of the intimate bilateral relationship between N. punctiforme and the isolate Agrobacterium tumefaciens Het4 using shotgun proteomics and microscopy uncovered a complex partnership characterized, among other traits, by competition for iron and facilitation for carbon. Although competitive interactions with A. tumefaciens Het4 compromise nitrogen fixation and stimulate the degradation of cyanophycin, mutualistic dependency prevails under inorganic carbon limitation. Both the absence of the high affinity bicarbonate uptake transporter SbtA and the prevalent extracarboxysomal localization of the carbon-fixing enzyme RubisCO, as detected by immunofluorescence microscopy, suggest that a weak carbon concentrating mechanism in N. punctiforme enforces a dependence on heterotrophic bacteria. Further, immunofluorescence, electron microscopic and proteomic analyses reveal a pronounced extracellular recycling of proteins under N- and C-limiting conditions. Our study shows that the pivotal influence of heterotrophic bacteria on symbiotic Nostoc strains should be considered when analyzing these cyanobacteria, especially in the free-living state. This work also sheds new light on how Nostoc benefits from the organic carbon provided by plant hosts.