Unraveling The Role Of GntR On The Regulation Of Alkane Hydroxylases AlkB₂ In Pseudomonas Aeruginosa DN1 Based On Transcriptome

As the major components of most crude oils,n-alkanes that represents a key environmental and economic issue is still a major challenge.Alkanes with different lengths were initial terminal oxidized through different alkane hydroxylases and initiate biodegradation.In Pseudomonas aeruginosa DN1,Alk B₂ was reported to play a key role on the alkane biodegradation,while the transcriptional regulator related alkB₂ is still unclesr.To deeply characterize the regulation mechanism of alkB₂ gene,this study used transcriptome analysis to identify the gene expression changes in wild-type strain and alkB₂mutant when grown in a n-alkane of C₂₀ containing environment.Meanwhile,gene knockout approach and RT-qPCR combined with EMSA experiments were performed to characterize the regulation mechanism of alkB₂ gene by putative transcriptional regulator.To analyze the gene expression changes in wild-type strain and alkB₂ mutant when grown in a n-alkane of C₂₀ containing environment,the RNA-Seq was performed via Illumina Novaseq platform in this study.The results showed 344 upregulated and 78 downregulated DEGs in alkB₂ mutant,compared to the wild-type strain DN1.KEGG pathway analyses revealed that most of the DEGs were associated with the pathway alkane metabolism,including MCPs related to chemotaxis,four flagellum assembly protein encoding genes,ong-chain fatty acid transportor Fad L encoding gene,iron carrier protein Fpt A enconding gene,and three transcriptional regulators encoding gene.Notably,the transcriptional regulator GntR（RS18845）located upstream of alkB₂ gene（RS18850）was upregulated.It is speculated that GntR has a certain regulatory effect on alkB₂.To investigate the effect of GntR on transcription of alkB₂,the gntR mutant was constructed,and growth curves of the strain DN1 and gntR mutant in the presence of 200mg/mL C₂₀ was determined based on the OD600.Compared with wide-type strain,the gntR mutant strain showed a markedly higher growth rate at the initial 24 h,while a similar growth profile was observed after 48 h cultivation.And the RT-qPCR showed that the expression of alkB₂ gene in the gntR mutant was independent on the presence of C₂₀,while the expression of alkB₂ gene was only induced by C₂₀ significantly in wild type DN1.These results demonstrated that GntR served as a repressor for alkB₂ expression,and n-alkane released its expression effect.On the basis of preliminary experiment,the expression and purification of GntR was performed to further investigate the direct interaction between GntR（RS18845）and the promoter region of alkB₂.Based on the EMSA,the DNA motif 5′-ATTGTCAGACAAT-3′was verified to be recognized by GntR regulator.However,the ability of DNA-binding was disrupted in the presence of C₂₀,suggesting that GntR displayed a perfect fusion of C₂₀ to regulate the expression of alkB₂.Amino acid sequence alignment of GntR in different strains showed that there was a conserved Arg49 in the GntR of DN1,which was site-directed mutated into Ala and electroporated into the gntR mutant.Compared to the the expression of alkB₂ of the wild type DN1,a similar phenomenon to the gntR mutant was seen in the defective strain in absence of Arg49,indicating that Arg49 played an important role in the binding of GntR to promoter.This report provides the insights into the importance of GntR in the regulation of degradation of alkane by alkB₂,and also provides important information for understanding the complex regulatory network of degradation of alkane.