The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135

Abstract

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3→NH2OH→N2), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR‐like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506‐bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH‐dnfR intergenic region and two binding sites in the dnfG‐dnfA intergenic region. Three binding sites of DnfR shared a 6‐bp repeated conserved sequence 5′‐GGTCTG‐N17‐GGTCTG‐3′ which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO‐1 to deal with the toxicity of ammonium

“…Both Alcaligenes ammonioxydans HO‐1 and A. faecalis JQ135 possess the ability of Dirammox. The dnfABC cluster in A. faecalis JQ135 contains key genes involving in the Dirammox process with DnfR serving as the positive transcriptional regulator (Xu, Qian, et al, 2022; Xu, Wang, et al, 2022). However, considering the huge taxonomic difference in Dirammox activity, further exploring into the function and molecular mechanisms of the dnf gene cluster in other Alcaligenes species like A. ammonioxydans HO‐1 in vivo is highly desired.…”

“…Moreover, recent studies have further refined the organization of dnf cluster in A. faecalis JQ135, including dnfABC and dnfDEFGHIR genes (Xu, Qian, et al, 2022). Gene clusters regulated by DnfR and dnfDEGI genes were predicted to be involved in nitrogen-related metabolism (Xu, Wang, et al, 2022). For example, DnfH is predicted to be a phosphoserine aminotransferase, while both DnfI and DnfG have been described as serine hydroxymethyltransferase.…”