Gene Function And Chemotaxis Of Pseudomonas Aeruginosa DN1 Involved In Petroleum Hydrocarbon Degradation

Petroleum hydrocarbon compounds pollution has drawn worldwide attention and bioremediation is considered to be a promising method.However,complex components and highly hydrophobic of hydrocarbon contaminants restricted the uptake and degradation of degrading bacteria.In this study,genes involved in petroleum hydrocarbon degradation and chemotaxis of pollutants in Pseudomonas aeruginosa DN1 were investigated to explore the mechanism how microorganisms uptake and transport hydrocarbons for bioremediation application.Previous studies showed that Pseudomonas aeruginosa DN1 had the ability to degrade crude oil,alkanes and aromatics.To further understand degradation mechanism,comparative genomics was used to analyze the alkane hydroxylase,ring hydroxylating dioxygenase and chemotaxis system in the six hydrocarbon-degrading bacteria.The results demonstrated that diverse degrading enzyme system existed in degradating bacteria,while the dissimilarity of gene homology and distribution affected degradation spectrum.Then degradation efficiency of crude oil and diverse alkanes of the DN1 by gas chromatography were detected,and the expression of alkane hydroxylase gene was analysed with RT-qPCR,genes function were further studied by constructing gene knockout mutants with homologous recombination technology.Results exhibited that the DN1 was capable of utilizing diverse n-alkanes with chain lengths of from 8 to 40 C atoms as the sole carbon source,and displayed high degradation rate?>85%?of crude oil and a majority of n-alkanes.RT-qPCR analysis showed that the five enzyme genes?alkB₁,alkB₂,p450,almA₁ and almA₂?could be induced by n-alkanes ranging from C₈ to C₄₀0 whereas indicated the dissimilarity of expression between those genes when grown on different n-alkanes.RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from C₈ to C₄₀0 whereas 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 were only upregulated in the presence of n-alkanes with chain lengths of 32C'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 to40 C atoms.AlkB₂ amino acid sequences of several degradative strains of Pseudomonas aeruginosa were compared and had homology of about 99.65%.Methyl-accepting chemotaxis proteins?MCPs?play a vital role in sensing external stimuli once bacterial chemotactic responses occur.Previous transcriptomic analysis demonastrated that some mcp genes of mcp04325,mcp10445,mcp28325,mcp29160 and so on,were up-regulated in the DN1 during fluoranthene degradation.On the basis of transcriptional analyses,mcp mutants were constructed to investigate the chemotactic responses of the DN1 to various aromatic hydrocarbons and alkanes and their intermediate metabolites.The isothermal titration calorimetry?ITC?was employed to examine the binding capacity.Results showed that the DN1 performed chemotactic responses to different hydrocarbons,it was hypothesized that the DN1's chemotactic responses involved in the degradation of hydrocarbons.The knockout of mcp00975,mcp04325,and mcp10445genes resulted in the declined chemotactic responses towards protocatechuic acid and some long-chain alkanes.The?mcp04325 related to declined chemotactic responses towards fluoranthene and pyrene,while?mcp00975 and?mcp04325 involved in naphthalene and phenanthrene.In addition,after stimultaneous knockout of mcp00975,mcp04325,and mcp10445 genes,the chemotactic responses towards protocatechuic acid significantly weakened.Compared to other mcp genes,mcp04325 gene played the dominant role,but ITC analysis illuminated that Mcp04325 protein hardly bonded to protocatechuic acid.Hence,it was speculated that although incapable of binding with protocatechuic acid,Mcp04325 protein could mediate chemotaxis.The underlying mechanism needs to be further explored.