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Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants., Gut bacteria are prevalent across insects including ants, but their precise roles are often unclear. Here, Hu et al. show that microbes aid ants by recycling nitrogen into bio-available amino acids. This function is conserved across the turtle ants, suggesting an ancient nutritional mutualism.

The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2 in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated. Bacteroids from FixJ and FixK2 mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1 mutant exhibit wild-type levels of hydrogenase activity. In b-galactosidase transcriptional assays with NifA and FixK2 expression plasmids, the FixK2 protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2 is involved in the transcriptional activation of symbiotic hydrogenase expression. By using b-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site. The slow-growing symbiont of the soybean plant, Bradyrhizobium japonicum, expresses a hydrogen uptake hydrogenase that oxidizes hydrogen under both free-living and symbiotic conditions. In the free-living state, the expression of the NiFe hydrogenase is regulated at the transcriptional level by hydrogen, oxygen, and nickel (21). These three signals exert their effects within a 50-bp region of DNA located between 99 and 149 bp upstream of the transcription start site of the hydrogenase structural genes (22). In addition, the hydrogenase promoter is s 54 dependent and requires integration host factor for full induction (5). The hoxA gene (32) is located approximately 12 kb downstream of the hydrogenase structural genes, in a region of the hydrogenase gene cluster previously shown to be necessary for free-living hydrogenase activity (23). The hoxA gene encodes a protein with extensive homology to transcriptional activators of hydrogenase expression in several other organisms, including HoxA in Alcaligenes eutrophus (12), HupR1 in Rhodobacter capsulatus (30), and HydG in Escherichia coli (31), all of which are members of the NtrC-like family of response regulators (17). Subsequent studies of the role of the HoxA protein in the biosynthesis of hydrogenase by our group (11) and another (33) have confirmed that HoxA is a transcriptional activator of hydrogenase expression under free-living, microaerobic conditions. Its cognate sensor protein is presently unknown. However, bacteroids from nodules formed by B. japonicum HoxA mutants exhibit wild-type levels of hydrogenase activity and extracts from Hup 1 bacteroids used in gel retardation assays