Regulation mechanism of the long-chain n-alkane monooxygenase gene almA in Acinetobacter venetianus RAG-1

As toxic pollutants, n-alkanes are pervasively distributed in most environmental matrices. Although the alkane monooxygenase AlmA plays a critical role in the metabolic pathway of solid long-chain n-alkanes (≥C₂₀) that are extremely difficult to degrade, the mechanism regulating this process remains unclear. Here, we characterized the function of AlmA in Acinetobacter venetianus RAG-1, which was mainly involved in the degradation of long-chain n-alkanes (C₂₆-C₃₈), among which, n-C₃₂ induced the almA promoter activity most. APR1 (AlmA Positive Regulator) that it is an AraC/XylS-type transcription regulator, a potential transcriptional regulator of almA, was screened by DNA-pull down, which was determined by conserved domain analysis. The deletion of apR1 severely reduced the capacity of strain RAG-1 to utilize long-chain n-alkanes (C₂₂-C₃₈), indicating the involvement of APR1 in n-alkanes degradation. The results of the APR1-dependent reporter system, electrophoretic mobility shift assay, and microscale thermophoresis further proved that APR1 was able to directly bind to the almA promoter region, thus activating the almA transcription. Furthermore, APR1 could inhibit self-expression through autoregulation in the absence of long-chain n-alkanes. n-C₃₂ acted as a ligand of APR1, and the amino acid residues Val10, Gln50, Ala99, and Ile106 at the N-terminus of APR1 were necessary for binding n-C₃₂. In addition, the key amino acid residues of APR1 within the C-terminal helix-turn-helix motif that bound to the downstream promoter region were confirmed by multiple sequence alignment and site-directed mutagenesis. The homologs of AlmA and APR1 shared a similar evolutionary course in the Proteobacteria; thus, this mode of regulation might be relatively conserved.

Importance: The extreme hydrophobicity of long-chain n-alkanes ({greater than or equal to}C₂₀) presents a significant challenge to their degradation in natural environments. It is, therefore, imperative to elucidate the regulatory mechanisms of the metabolic pathways of long-chain n-alkanes, which will be of great significance for the future application of hydrocarbon-degrading bacteria to treat oil spills. However, the majority of current studies have focused on the regulatory mechanisms of short- and medium-chain n-alkanes, with long-chain n-alkanes receiving comparatively little attention. In this study, we identified APR1, a transcriptional regulator of the alkane monooxygenase AlmA in Acinetobacter venetianus RAG-1, and characterized its function and regulatory mechanism. In the presence of ligand n-C₃₂, APR1 could directly activate the transcription of almA, which was involved in the n-C₃₂ metabolism. The amino acid residue unique to the C-terminal DNA-binding domain of AraC/XylS type n-alkanes transcription regulators was also identified. These findings further improved our understanding of the long-chain n-alkanes degradation mechanism, which is important for the management of petroleum pollution.