Molecular Mechanism Study On CrgA To Regulate The Degradation Of Medium-To-long Chain Alkanes In Pseudomonas SJTD-1

The method of microbial remediation is considered to be an effective solution to oil pollution because it's green and energy saving.Up to now,a variety of petroleum degradation strains,degradation pathways and related enzyme systems have been isolated and identified,and some progress has been made in the regulation of degradation.However,as the difficult point,the regulation mechanism of middle-to-long chain alkanes degradation is still unclear.Our team previously isolated a strain that can efficiently degradate alkanes of C12-C32 and belongs to Pseudomonas aeruginosa named SJTD-1.Through the whole genome sequencing and annotation,proteomic analysis methods,we selected some potential alkane degradation genes:P450_1?P450₂?alkB1?alkB2?almA1?almA2?ladA1?ladA2?etc.,and alkB2 gene has been proved to participate in medium-to-long chain alkane degradation in SJTD-1.Through the pull-down experiment,we screened out some transcriptional regulators including predicted CrgA that may participate in the degradation and metabolism of alkanes in SJTD-1 strain.This work aims to explore the possible regulatory founction of the transcriptional regulator CrgA protein on several medium-to-long chain alkane-degradation genes,especially the alkB2 gene both in vivo?exogenous expression of CrgA protein,EMSA,Footpriting and dissociation experiment?and in vitro?gene knockout,growth curve measurement,RT-qPCR and fluorescence experiment?.Through multiple sequence alignment,evolutionary tree analysis and simulation of tertiary structure,the predicted CrgA protein in SJTD-1 strain shows high homology and sequence similarity with CrgA protein in Neisseria meningitidis,so it's classified to CrgA subfamily of LysR family transcriptional regulator;EMSA showed that CrgA protein could specifically bind to the upstream promoter region of alkB2,almA2,ladA1,ladA2 genes and crgA gene.Through Footpriting,we found when CrgA protein binded to promoter region of alkB2 gene,there were two binding regions with mirror structure?Imperfect Inverted Repeat,IIR?:5'-CCCTTGGCCGGC/CTGCCCCATCCC-3'and 5'-TCGTTCTGT/CCGCCGCT-3'.Binding was significantly weakened after the structure was cut off in half and the reverse symmetric structure was destroyed.It showed the importance of mirror structure to the combination.Alkane intermediate metabolites,16 acyl CoA and 18 acyl CoA could release the binding between CrgA protein and promoter region of alkB2 upstream,however,acetyl CoA have no this effect.Through growth experiment,we found that crgA gene knockout accelerated the growth rate of the strain when alkane as the sole carbon source.SJTD-1 and crgA knockout strain S1?crgA were cultivated in glucose and different long chain alkane training?C12-C24?,and compared with cultivated in the glucose,when cultivated in the alkanes,the alkB2 gene transcription expression increased significantly.When the Gene knockout crgA is also under the presence of alkanes,alkB2 gene transcription level in S1?CrgA was higher.The same result was applied to almA2?ladA1?ladA2 gene;In fluorescence experiment we found that green fluorescent report plasmid with alkB2 promoter in S1?crgA strains,the fluorescent protein expression was higher than wild type strain SJTD-1 with same plasmid.The expression of green fluorescent protein was higher in the presence of alkanes than glucose.In summary,we concluded that CrgA can participate in the alkanes degradation regulation process of SJTD-1 strain by inhibiting the transcription of alkB2 gene.Moreover,CrgA protein may also be involved in the transcriptional regulation of almA2,ladA1 and ladA2 genes,which can encode long-chain alkane hydroxylases.This work was a further advance on the research of molecular level regulation mechanism of alkane degradation and also provides reliable theoretical support for the subsequent modification to highly efficient and applicable strains.