Study On The Transcriptional Regulation Of 3-phenoxybenzoate Microbial Catabolism

3-Phenoxybenzoate(3-PBA)is the main metabolite of pyrethrins pesticides degradation in bacterium.It is also the derivant of diphenyl ethers compounds,which have a stable chemical structure.Research on the degradation of 3-PBA will contribute significantly to understand the intact pathway and mechanism of pyrethrins pesticide's biodegradation,and provide the theory base for the bioremediation of pyrethrins' restudies in the environment.In the previous studies in our lab,a pyrethrins-degrading bacterium Sphingobium wenxiniae JZ-1T was isolated.The pyrethroid-hydrolyzing carboxylesterase gene pytH and 3-PBA 1',2'-dioxygenase gene cluster pbaA1A2BC were cloned successively.PytH catalyzes pyrethrins to permethric acid and 3-PBA,and this gene is expressed constitutively.pbaA1A2BC encodes an angular dioxygenase which catalyzes 3-PBA to 3-hydroxybenzoic acid and catechol,which are further degraded by other reported genes.Up to now,the key genes involved in the biodegradation of pyrethrins had been all coloned.At the same time phaA1A2B was characterized as inducible expression gene cluster by its substrate 3-PBA,which indicated that a mechanism of transcriptional regulation was present in JZ-1T.This study focuses on the molecular mechanism of transcriptional regulation of 3-PBA's catabolism.The results of this paper will improve our understanding of the 3-PBA degradation progress in bacterium,and improve the application efficiency of degrading strain.At the same time,this study will expand the understanding of the transcription mode of bacterial catabolism of pyrethrins pesticide.1.Identification of the transcriptional activator PbaR of pbaA1A2B gene cluster in Sphingobium xenxiniae JZ-1.Transcriptional unit of phaA1A2BC in strain JZ-1T was studied,and the results showed that pbaAl,pbaA2 and pbaB are all in one transcriptional unit while pbaC is in another dependent transcriptional unit.Base "A" in 48-bases to the initiation codon was identified as the transcription start site of pbaA1A2B cluster,and base "C" in 33-bases to the initiation codon was identified as the transcription start site of pbaC.DNA elements including-10 box,-35 box and ribosome binding site were predicted in the two promoters.Pomoter DNA of pbaA1A2B cluster was amplified and used for affinity purification of promoter specific binding proteins,4 SDS-PAGE protein bands whose molecular mass are less than 35 kDa were identified by the MALDI-TOF/TOF mass spectrometry.After comparing in genome of JZ-1T,an IclR family transcriptional regulator was proposed as the target transcription factor,and it was named as PbaR.When gene pbaR was knockout,corresponding strain JZMT lost the capacity of 3-PBA utilization while genetic complementation of pabR recovered JZMT to the wild phenotype.RT-qPCR experiment showed that pbaAl,pbaA2 and pbaB's transcription level of mutant stain was much lower than the wild type in the same culture conditions as well as pbaR complementary strain.However,knockout of pbaR had none significant influence on the transcription of pbaC gene.PbaR was heterologously expressed in Escherichia coli BL21(DE3),and purified protein could bind to the promoter DNA of pbaA1A2B but not the pbaC promoter's probe.The formation of DNA-protein complex is 3-PBA independent.DNase I footprinting showed that PbaR binds to a 29-bp DNA motif in the pbaA1A2B promoter,and a 10-bp palindrome sequence in this motif was proved to be important for PbaR recognition.Another 19-bp sequence,which around the 10-bp palindrome sequence,was proved to be important for the stability of DNA-protein complex formation. PbaR has some special regulation mode comparison with other reported IclR-type transcriptional regulators.Firstly,the binding site of PbaR is located between the transcription start site and the initiation codon,which is usually a binding site of repressors,but PbaR is a transcriptional activator.Secondly,only one palindrome sequence exists in the binding motif,while other IclR-type regulators bind with 2-3 repeat motifs.Finally,sequence around the palindrome sequence is important for the stability of regulator binding.These characteristics of PbaR increase the variety of regulatory model in the IclR-family of transcriptional regulators.2.Plasmids for genome shotgun screening of transcriptional regulator genes involved in the bacterial xenobiotic catabolism.A 3-PBA degradation bacterium,Sphingobium sp.TP316,was isolated in this study,and relative genes such as pbaA1A2B and pbaR shared 100%similarity with those founded in the strain JZ-1T.A batch culture phenomenon was found in the present of 3-PBA and fructose,which suggested that a mechanism of carbon catabolite repression would regulate the catabolism of 3-PBA in TP316.It was proposed that pbaR was regulated by another transcription factor which probably was a global regulator.However,DNA affinity test had not achieved the prospective goal.To search for the proposed transcriptional regulator,a positive library screening plasmid pSRGFP-18 and its series plasmids were constructed.Principle of the screening plasmid is based on regulation relationship between catabolic genes and their transcriptional regulators.A reporter gene egfp was used,when promoters of catabolic genes were linked with egfp,and corresponding plasmid could be used for genome library screening.When evaluated by reported activation type regulation system benR-benABC and repression type regulation system nicR-nicC in Pseudomonas putida KT2440,pSRGFP-18 plasmid worked well which indicated that it was suitable for transcriptional regulator gene screening of known catabolic genes.3.The carbon catabolite repression of 3-PBA degradation might be mediated by CbpR which regulates the transcription of pbaR gene.By using the constructed plasmid pSRGFP-18,genome library was screened for the transcriptional regulator of pbaR gene,and a positive clone 515-23 was found.Sequence analysis showed that a Crp/FNR family transcriptional regulator existed in the insertion fragment,which was named as cbpR meaning cAMP binding protein regulator.When cbpR was knockout,the transcription of pbaR gene lost correlationship with the present of fructose and its transcription level had no significant difference in various culture conditions.Complementation of cbpR recovered the repression phenomenon of pbaR by fructose in mutant strain.Transcription level of pbaA2 represented the same changes to pbaR.These results indicated that cbpR encoded a transcriptional activator to pbaR,which responsed to the absent of the preferential carbon resource fructose.EMSA showed that CbpR could bind with pbaR promoter DNA probes,when cAMP was absent,the binding complex was in an oligomerization state and cAMP adding altered CbpR-DNA complex to multimer.According to these results,it was proposed that none cAMP binding CbpR acted as a repressor while cAMP binding CbpR acted as an activator. DNase I footprinting showed that a 23-bp DNA motif in pbaR promoter was the binding site of CbpR,and this motif shared high similarity with binding site of CRP,which was a homologous protein of CbpR in E.coli.According to the results above,the cAMP binding protein CbpR acted a transcriptional regulator of pbaR gene in response to the present of preferential carbon resources.When the best carbon resource fructose was abundant,concentration of cAMP was relative low in cells,and none cAMP binding CbpR acted as a repressor to pbaR.When preferential carbon resources were deficient,concentration of cAMP increased and cAMP-CbpR active the transcription of pbaR.PbaR then actives the transcription of pbaA1A2B in response to the present of 3-PBA.This mechanism explained the reason of carbon catabolite repression of 3-PBA degradation by fructose.