Unravelling the role of GntR on the regulation of alkane hydroxylase AlkB2 in Pseudomonas aeruginosa DN1 based on transcriptome analysis

Abstract

Aims: The purpose of this study is to acquire a comprehensive understanding of the involvement of the gene alkB₂ in alkane degradation.

Methods and results: The changes of gene expression in the wild-type and alkB₂ knockout strains of Pseudomonas aeruginosa DN1 were characterized based on transcriptional profiling, when grown in a medium containing eicosane (C₂₀ n-alkane) as the sole carbon source. Compared to wild-type, approximately 7% of the genes in the knockout mutant was significantly differentially expressed, including 344 upregulated genes and 78 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that numerous differentially expressed genes (DEGs) were potentially associated with degradation or physiological response to n-alkane, including genes encoding methyl-accepting chemotaxis proteins (MCPs), an outer membrane fatty acid transport protein (FadL), a membrane receptor protein (FptA), oprin and transcriptional regulators. Notably, the transcriptional regulator gene gntR (RS18845) located upstream of alkB₂ (RS18850) was upregulated. The possible regulatory function of this transcriptional regulator on alkB₂ was investigated using a gene knockout approach and quantitative reverse transcriptase PCR (RT-qPCR) combined with electrophoretic mobility shift assay (EMSA) experiments. The RT-qPCR results showed that in the gntR mutant, alkB₂ expression was independent of the presence of eicosane, while its expression was significantly induced by the substrate when GntR was produced. Based on the EMSA analysis, the palindromic DNA motif 5'-ATTGTCAGACAAT-3' was verified as being recognized by GntR, and two copies of GntR were able to bind this sequence. However, the interaction between GntR and DNA was altered in the presence of eicosane, suggesting that GntR could bind with eicosane to regulate the expression of alkB₂ .

Conclusion: These findings indicate that GntR plays a key role in the transcriptional regulation of alkB₂ , which affects the degradation of C₂₀ n-alkane in P. aeruginosa DN1.

Significance and impact of the study: This report presents insights into the significance of GntR in the regulation of alkane degradation by alkB₂ , and increases our understanding of the complex regulatory network involved in alkane degradation.

Keywords: Pseudomonas aeruginosa; AlkB2; RNA-seq; n-alkane degradation; transcriptional regulator GntR.