Dual Regulation of the <i>allF</i> Operon by ArcA and AllS Enables Anaerobic Allantoin Utilization in <i>Escherichia coli</i>

During anaerobic growth, <i>Escherichia coli</i> is capable of utilizing allantoin as its sole nitrogen source. Allantoin, a purine derivative, is initially degraded into ureidoglycolate with the release of two NH₃. Ureidoglycolate can then enter either the glycerate branch (to 2-phosphoglycerate) or the oxamate branch (releasing additional NH₃). In the oxamate branch, ureidoglycolate first is oxidized to oxalurate, then converted to oxamate and carbamoyl phosphate by oxamic transcarbamylase (OXTCase); carbamoyl phosphate, in turn is used by carbamate kinase (CK) to generate ATP and NH₃. This study focuses on the transcriptional regulation of OXTCase and CK, which catalyze the final two steps of the oxamate branch and are encoded by the <i>allFGHK</i> operon (<i>allF</i> operon), the most recently identified genes in the allantoin pathway. Transcription of the <i>allF</i> operon was analyzed using a plasmid-borne <i>allF'-'lacZ</i> reporter and relevant regulator mutants. High expression of the <i>allF</i> operon under anaerobiosis with allantoin requires the local regulator AllS and the global regulator ArcA. EMSA confirmed the direct binding of AllS and ArcA to the promoter of <i>allF</i>. These findings indicate that the oxamate branch is directly regulated by the activator AllS, one of two local regulators (AllR and AllS) in the allantoin pathway. Furthermore, we identified ArcA as the activator of <i>allF</i> operon transcription under anaerobic conditions. Although allantoin degradation is known to occur only anaerobically, the regulator remains unidentified. Our findings demonstrate ArcA's involvement. ArcA, the regulator of the <i>allF</i> operon, may also control other anaerobic genes in the allantoin cluster, directly or indirectly.