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

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.

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n-alkane pollution by Pseudomonas aeruginosa DN1.

AIMS: The purpose of this study was to elucidate the characteristics of multiple alkane hydroxylase systems in Pseudomonas aeruginosa DN1, including two homologues of AlkB (AlkB1 and AlkB2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA1 and AlmA2 ). METHODS AND RESULTS: DN1 was capable of utilizing diverse n-alkanes with chain lengths from 8 to 40 C atoms as the sole carbon source, and displayed high degradation efficiency (＞85%) of crude oil and a majority of n-alkanes using gas chromatography method. RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from medium-chain length to long-chain length which indicated the dissimilarity of expression between those genes when grown on different n-alkanes. Notably, the expression of alkB2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C20 and C32 . Meanwhile, long-chain n-alkanes (C20 -C36 ) significantly elevated cyp153 expression level, and the expression of two almA genes was only upregulated in the presence of n-alkanes with chain lengths of 20C's and longer. Furthermore, the disruption of those genes demonstrated that AlkB2 appeared to play a key role in the biodegradation of substrates of a broad-chain length ranges, besides other alkane hydroxylase systems ensured the utilization of n-alkanes with chain lengths of from 20 to 40 C atoms. CONCLUSION: The five functional alkane hydroxylase genes make DN1 an attractive option for its versatile alkane degradation, which is primarily dependent on the expression of alkB2 . SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that P. aeruginosa DN1 is a predominately potential long-chain n-alkane-degrading bacterium with multiple alkane hydroxylase systems in crude oil-contaminated environment.

CrgA Protein Represses AlkB2 Monooxygenase and Regulates the Degradation of Medium-to-Long-Chain n-Alkanes in Pseudomonas aeruginosa SJTD-1.

AlkB monooxygenases in bacteria are responsible for the hydroxylation of medium- and long-chain n-alkanes. In this study, one CrgA protein of Pseudomonas aeruginosa SJTD-1, a member of LysR family, was proved to regulate AlkB2 monooxygenase and the degradation of medium-to-long-chain n-alkanes (C14-C20) by directly binding to the upstream of alkB2 gene. Two specific sites for CrgA binding were found in the promoter region of alkB2 gene, and the imperfect mirror repeat (IIR) structure was proved critical for CrgA recognition and binding. Hexadecyl CoA and octadecyl CoA could effectively release the CrgA binding and start the transcription of alkB2 gene, implying a positive regulation of metabolic intermediate. In the presence of medium-to-long-chain n-alkanes (C14-C20), deletion of crgA gene could enhance the transcription and expression of AlkB2 monooxygenase significantly; and in n-octadecane culture, strain S1ΔalkB1&crgA grew more vigorously than strain S1 ΔalkB1 &crgA . Almost no regulation of CrgA protein was observed to alkB1 gene in vitro and in vivo. Therefore, CrgA acted as a negative regulator for the medium-to-long-chain n-alkane utilization in P. aeruginosa SJTD-1. The work will promote the regulation mechanism study of n-alkane degradation in bacteria and help the bioremediation method development for petroleum pollution.

In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes.