# Dual Regulation of the allF Operon by ArcA and AllS Enables Anaerobic Allantoin Utilization in Escherichia coli

## TL;DR

This paper explains how Escherichia coli uses allantoin for nitrogen under anaerobic conditions by regulating specific genes with two key regulators, AllS and ArcA.

## Contribution

The study identifies ArcA and AllS as dual regulators of the allF operon, enabling anaerobic allantoin utilization in E. coli.

## Key findings

- The allF operon is activated by AllS and ArcA under anaerobic conditions with allantoin.
- EMSA confirmed direct binding of AllS and ArcA to the allF promoter.
- ArcA is shown to be a key regulator of anaerobic allantoin metabolism in E. coli.

## Abstract

During anaerobic growth, Escherichia coli 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 allFGHK operon (allF operon), the most recently identified genes in the allantoin pathway. Transcription of the allF operon was analyzed using a plasmid-borne allF′–′lacZ reporter and relevant regulator mutants. High expression of the allF 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 allF. 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 allF 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 allF operon, may also control other anaerobic genes in the allantoin cluster, directly or indirectly.