| Dibenzofuran（DBF）is a heterocyclic compound.It is an aromatic compound with two benzene rings bonded to a central furan ring.The carbon atoms are bonded to hydrogen atoms.It is a white solid,volatile compound that is soluble in organic solvents.DBF is used as a model compound for the degradation of dioxins.DBF is a component of coal tar,creosote and crude oil.Due to the long half-life,strong toxicity,and serious effects of DBF,the cleaning of sites polluted with these type of contaminants is a severe problem.Furthermore,they can cause mutagenic,carcinogenic and other effects in living organisms.Several efforts were made to develop effective bioremediation methods,and some efficient physicochemical approaches were developed for DBF removal.Though,the effective field application of such techniques is not possible due to the persistence of DBF in the environment.As an effective method,the naturally occurring degradation of these contaminants has acquired great importance within remediation studies.The degradation of DBF involves two main pathways:angular and lateral dioxygenation.The angular pathway,initiates when a molecular oxygen attacked the 4,4a carbon atoms and produces an unstable hemiacetal.This cis-dihydrodiol cleaved suddenly to yield 2,3,2-trihydroxybiphenyl（THBP）.Then,the ring is then broken to produce salicylic acid,which is then transformed to gentisic acid or catechol.On the other hand,lateral degradation is occurring mostly in biphenyl and naphthalene biodegradation.In the lateral dioxygenation,the two adjacent carbon atoms are attacked at positions 1-4.Biodegradation of DBF by microorganisms is an effective approach for the treatment of DBF contamination,and therefore it is important to understand the degrading mechanism of the microorganisms.In the current study,we isolated a new strain Pseudomonas aeruginosa FA-HZ1 from the landfill waste water,which can efficiently degrade DBF by both meta cleavage and angular dioxygenase pathway.Firstly,a novel strain of P.aeruginosa,FA-HZ1,with the efficient capability to use DBF as a sole source of carbon and energy,was isolated from the landfill waste water.The complete genome of P.aeruginosa FA-HZ1 was sequenced with the Pacific Biosciences（PacBio）RS II sequencer（Pacific Biosciences,USA）.The whole genome sequence was further analyzed with RAST and NCBI,the total size of the genome is 6,838,112 bp consist of one circular chromosome with a G+C content of 66%.The genome also encodes 4 ncRNAs,12 rRNAs,and 66 tRNAs,which make up 0.001%of the genome.A total of 6,064 CDS were identified in the genome of FA-HZ1.Through the RAST server,annotation,158 genes were identified that were involved in polycyclic aromatic compounds degradation.Furthermore,the subsystem features contain 373genes related with“cofactors,vitamins pigments and prosthetic groups,353 with membrane transport,122 genes were associated with sulfur metabolism 233 with RNA transport,310 with protein metabolism and some were associated with virulence,disease and defense etc.The whole genome sequencing and genome-based functional analysis of P.aeruginosa FA-HZ1 deliver us with deep understandings into its genetic basis for metabolisms of aromatic compounds,DBF degradation,biosynthesis of secondary metabolites,antibiotic resistance,bacterial responses to environmental conditions,cell motility and chemotaxis,membrane transport,secretion and vascular transport etc.The obtained genome data play a key role to develop rational strategies for biodegradation of DBF polluted environments.The results also provide a useful reference for the genomics,proteomics and transcriptomics of other microorganisms.Secondly,the conditions for novel strain Pseudomonas aeruginosa,FA-HZ1,with high DBF-degrading activity were optimized.The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30°C,pH 5.0,rotation rate of 200 rpm and 0.1 mM DBF as a carbon source.The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied.The new strain was positive for arginine double hydrolase,gelatinase and citric acid,while it was negative for urease and lysine decarboxylase.It could utilize citric acid as its sole carbon source,but was negative for indole and H2S production.Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran,1,2-dihydroxydibenzofuran,2-hydroxy-4-（3′-oxo-3′H-benzofuran-2′-yliden）but-2-enoicacid,2,3-dihydroxybenzofuran,2-oxo-2-（2′-hydrophenyl）lactic acid,and 2-hydroxy-2-（2’-hydroxyphenyl）acetic acid were detected and identified through liquid chromatography-mass analyses.FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways.A novel enzyme“HZ6359 dioxygenase”,was amplified and expressed in pET-28a in E.coli BL21（DE3）.The recombinant plasmid was successfully constructed,and was used for further experiments to verify its function.In addition,the strain FA-HZ1 can also degrade halogenated analogues such as 2,8-dibromo dibenzofuran and4-（4-bromophenyl）dibenzofuran.Undoubtedly,the isolation and characterization of new strain and the designed pathways is significant,as it could lead to the development of cost-effective and alternative remediation strategies.The degradation pathway of DBF by P.aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.The identification of DBF-degradation intermediates suggests that strain FA-HZ1 utilizes the lateral deoxygenation and meta-cleavage pathways to degrade DBF.These results indicate that strain FA-HZ1 can degrade DBF efficiently.Thirdly,the HZ6359 is identified as the DBF catalyzing protein expressed by the bacterial gene HZ6359 by in vitro analysis.As this protein is identified for the first time in our lab,therefore,the crystallographic study for this actively used protein is not available.Due to the unavailability of co-crystal structure in online database,the computational tools were used to get the 3D structure for the protein and further used for the structural and dynamic study of the protein in the presence and absence of the substrate.The obtained structure was found similar to the available structure of the Acireductone dioxygenase,from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalysing different reactions with the same substrates（acireductone and O2）depending upon the metal bound in the active site.Our modelled structure consists of beta parallel and anti-parallel sheets and alpha helices connected by loops.Further,the dynamic simulation studies state that substrate required readjustment to stabilize itself in large pocket.This computational study would provide a base for the researchers to further elaborate the structure-activity relationship of the protein and substrate complex.Fourthly,Proteomics has become one of the main methods to analyse and understand various function of genes.However similar to other analysis approaches,the demonstration of the huge amounts of acquired data in simply interpretable ways remains hard.The unique challenges and present solutions for analysing various characteristics of proteomics data,from the spectra through proteins identification to pathway analysis,are explained.We provide our ideas about upcoming methods for proteomics data analysis.The strain FA-HZ1 was grown with DBF as the main carbon source（Experimental group）as well as with glycerol（Controlled group）.The samples were collected during exponential phase from both the groups and sent for further analysis.A total of1459 proteins were identified in strain FA-HZ1 in which 100 were up regulated while 104 proteins were down regulated.Different genes related to various functions such as HZ6359,HZ772,HZ5387,HZ5952,HZ5519,HZ325 dioxygenases,HZ4256,HZ4257,HZ4506,HZ4254,HZ4121,HZ5342,HZ5669,HZ176,HZ4256,HZ896,HZ4011,HZ3013,HZ3320,HZ899,HZ521,HZ5340,HZ4492 dehydrogenases as well as decarboxylase HZ2801 and many other proteins involved in other functions such as cellular metabolism,chemo taxis and motility etc.were also identified.Fifthly,The PAHs persistence and lethality in the environments have resulted in many severe complications.These toxic compounds need to be removed from the environment so,the researchers have focused on the capability of various types of microorganisms（bacteria and fungi）to degrade these toxic polycyclic aromatic compounds into harmless or less toxic compounds.In the present study a microbial consortium consisting of about six strains including Pseudomonas puta B6-2,Pseudomonas putida KT2440,Methylobacterium sp.strain DM1,FA-HZ1,Stenotrophomonas strain.Ste-HZ1 and Achromobacter sp.was developed to check the degrading efficiency of various carbon sources such as NAP,BP,DBF,CAR and phenol.The consortium showed maximum growth as well as degradation rate as compared to other strains.Among all the PAHs used in this study naphthalene was highly degraded by both consortium and other single strain such as strain B6-2,KT2440 and DM1 but the degradation efficiency of consortium was maximum and effective,while carbazol was the least degraded compound by both consortium as well as other strains.Though,further research is required to understand what exactly happening in PAH-polluted environments and how these microbial communities can play their important role to get rid of these unwanted and toxic compounds from the environments.Finally,a novel strain Pseudomonas aeruginosa strain FA-HZ1 was isolated from the landfill waste water.The strain FA-HZ1 can efficiently degrade DBF into various intermediates by both angular and dioxygenation pathway which only occurs in biphenyl and naphalene utilizing bacteria.DBF biodegradation by strain Pseudomonas aeruginosa was never reported before so,this work highlights DBF degrading efficiency of Pseudomonas aeruginosa strain FA-HZ1 for the first time. |
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