Canavanine utilization and detoxification in bacteria

Abstract

The nitrogen rich compound guanidine is common in nature. A variety of molecules contain a guanidino group, ranging from amino acids like arginine and canavanine to the nucleobase guanine and secondary metabolites like streptomycin. Guanidine is sensed by at least four different classes of riboswitches that are widespread in bacteria. However, limited insights into the source and physiological role of guanidine in nature exist. For this thesis, canavanine, the -oxa-analogue of arginine, was explored as a source of guanidine. Thereby, a novel canava-nine-degrading Pseudomonad was isolated and due to its ability to grow on canavanine as sole carbon and nitrogen source it was named Pseudomonas canavaninivorans. Within this work the bacterium is characterized employing polyphasic taxonomy and screened for its spe-cific phenotypic traits that allow distinguishing it from closely related type strains. Furthermore, it is presented how the bacterium circumvents the toxicity of canavanine, which originates from incorporation into proteins as well as arginase-mediated hydrolysis to canaline that forms sta-ble oximes with carbonyls. First, a specific canavanine degradation pathway is described in great detail. The key enzyme, a PLP-dependent canavanine--lyase, was subjected to exten-sive characterization and further enzyme activities that facilitate canavanine utilization are elu-cidated. In addition, the distribution of the novel pathway and its implications in nature are dis-cussed. Second, it is presented how P. canavaninivorans achieves error-free translation. The arginine-tRNA-synthetase of the bacterium is not able to discriminate between arginine and canavanine as substrates, so it uses a standalone specific canavanyl-tRNAArg editing protein to protect itself from misincorporation. We therefore propose canavanyl-tRNAArg deacylase (CtdA) as the systematic name for the editing protein. The results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in na-ture. Finally, guanidine riboswitch-associated gene functions are explained in order to get in-sights into the compound’s physiology and purpose. Not only is it shown that guanidine can be used as a nitrogen source but also guanidine riboswitches are often associated with the newly described CtdA. Beyond being used as a source of nitrogen, the findings hint towards an in-trinsic connection between canavanine and guanidine metabolism. In sum, the data suggests a paradigm shift from riboswitch-controlled detoxification of guanidine to the uptake and assimila-tion of this enigmatic nitrogen-rich compound and to its usage as an indicator molecule for guanidine-group containing molecules like canavanine.