Characterization of guanidine carboxylases.

Abstract

Guanidine metabolism has been an overlooked aspect of the global nitrogen cycle until RNA sensors (riboswitches) were discovered in bacteria that bind the nitrogen-rich compound. The associated genes were initially proposed to detoxify guanidine from the cells. We were intrigued by a genetic organization where the guanidine riboswitch is located upstream of an operon comprising a carboxylase, two putative hydrolases, and an assigned allophanate hydrolase. An ABC transporter is located on the same operon with a periplasmic binding domain that is indicative of an importer. Therefore, we hypothesized that certain bacteria actively import guanidine and assimilate the nitrogen. To test this hypothesis, we searched for bacteria that were able to assimilate guanidine. We isolated three enterobacteria (Raoultella terrigena str. JH01, Erwinia rhapontici str. JH02 and Klebsiella michiganensis str. JH07) that utilize guanidine efficiently as a nitrogen source. Proteome analyses demonstrate that the expression of the guanidine riboswitch-associated carboxylase, in conjunction with associated hydrolases and transport genes, is markedly elevated in the presence of guanidine. Subsequent analysis of the carboxylases that are homologous to urea carboxylase confirmed the substrate preference of guanidine over urea. This chapter outlines a procedure for the isolation of guanidine-assimilating bacteria and the analysis of their proteome to identify enzymes responsible for guanidine degradation. Finally, an assay for the characterization of the endogenous guanidine carboxylases in comparison with the endogenous urea carboxylase from E. rhapontici is described.