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 (AlkB 1 and AlkB 2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA 1 and AlmA 2 ). 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 alkB 2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C 20 and C 32 . Meanwhile, long-chain n-alkanes (C 20 -C 36 ) 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 AlkB 2 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 alkB 2 . 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.

Stenotrophomonas maltophilia is a species with immensely broad phenotypic and genotypic diversity that could widely distribute in natural and clinical environments. However, little attention has been paid to reveal their genome plasticity to diverse environments. In the present study, a comparative genomic analysis of S. maltophilia isolated from clinical and natural sources was systematically explored its genetic diversity of 42 sequenced genomes. The results showed that S. maltophilia owned an open pangenome and had strong adaptability to different environments. A total of 1612 core genes were existed with an average of 39.43% of each genome, and the shared core genes might be necessary to maintain the basic characteristics of those S. maltophilia strains. Based on the results of the phylogenetic tree, ANI value and the distribution of accessory genes, genes associated with the fundamental process of those strains from the same habitat were found to be mostly conserved in evolution. Isolates from the same habitat had a high degree of similarity in COG category, and the most signi cant KEGG pathways were mainly involved in carbohydrate and amino acid metabolism, indicating that genes related to essential processes were mostly conserved in evolution for the clinical and environmental settings. Meanwhile, the number of resistance and e ux pump gene was signi cantly higher in the clinical setting than that of in the environmental setting. Collectively, this study highlights the evolutionary relationships of S. maltophilia isolated from clinical and environmental sources, shedding new light on its genomic diversity.

Aims:The purpose of this study is to acquire a comprehensive understanding of the involvement of the gene alkB 2 in alkane degradation. Methods and Results:The changes of gene expression in the wild-type and alkB 2 knockout strains of Pseudomonas aeruginosa DN1 were characterized based on transcriptional profiling, when grown in a medium containing eicosane (C 20 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 alkB 2 (RS18850) was upregulated. The possible regulatory function of this transcriptional regulator on alkB 2 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, alkB 2 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 alkB 2 . Conclusion:These findings indicate that GntR plays a key role in the transcriptional regulation of alkB 2 , which affects the degradation of C 20 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 alkB 2 , and increases our understanding of the complex regulatory network involved in alkane degradation.