CycA-Dependent Glycine Assimilation Is Connected to Novobiocin Susceptibility in Escherichia coli

Escherichia coli serine hydroxymethyltransferase (GlyA) converts serine to glycine, and mutants are auxotrophic for glycine. CycA is a transporter that mediates glycine uptake. Deleting in E. coli strain W3110 led to activation of CysB, which was related to novobiocin (NOV) susceptibility. Moreover, deleting resulted in increased sensitivity to NOV, and this could be reversed by high concentrations of glycine. Reverse mutants of Δ were selected and one of them had a mutation in , the gene encoding threonylcarbamoyl-AMP synthase. Subsequent proteome analysis showed that deleting led to increased expression of TcyP and TdcB, making this bacterium dependent on CycA for glycine assimilation. Furthermore, deleting in a Δ background caused a severe growth defect on Luria-Bertani medium, which could be complemented by high concentrations of exogenous glycine. Mutation of led to decreased expression of TdcB but increased expression of ThrA/B/C and LtaE, which favored the conversion of threonine to glycine and thus avoided the dependence on CycA. Correspondingly, deleting of , , or could reverse the NOV-sensitive phenotype of Δ mutants. Overexpression of resulted in increased sensitivity to NOV, whereas deleting this gene caused NOV resistance. Moreover, overexpression of led to increased accumulation of NOV upon drug treatment. Therefore, inactivation of in E. coli led to CycA-dependent glycine assimilation, which enhanced the accumulation of NOV and then made the bacterium more sensitive to this drug. These findings broaden our understanding of glycine metabolism and mechanisms of NOV susceptibility. Novobiocin (NOV) has been used in clinical practice as an ATPase inhibitor for decades. However, because it has been withdrawn from the market, pharmaceutical companies are searching for other ATPase inhibitors. Thus, probing the mechanisms of susceptibility to NOV will be beneficial to those efforts. In this study, we showed that inactivation of in E. coli led to CycA-dependent glycine assimilation, which accompanied the accumulation of NOV and thereby increased the sensitivity to this drug. To date, this is the first report demonstrating the linkage between glycine assimilation and NOV susceptibility, and it is also the first report showing that YrdC is able to modulate the metabolic flux of threonine.