Screening Of Carbazole Degrading Bacteria And Cloning, Characterization Of Meta-Cleavage Dioxygenases

We have collected 4 samples of wastewa ter and soil from Jilin Chemica l Pla nt, SipingChemica l Pla nt, Ginsber Brewery Group and Jia ngsu Chemica l Pla nt. A total of 12 bacteriumstrains which could use carbazole as the sole carbon, nitrogen and energy source were isola tedand all these isoa ltes were Gram-negative. Based on 16S rDNA sequence analysis , strainsJH061, PJ062, JS061 and JS062; JH051, PJ051, PJ052 and LH051; JH052; JH062; PJ053 andPJ061 were identified as Sphingomonas sp.；Bacillus sp.；Chryseobacterium sp.;Hydrogenophaga intermed ia ; Arthrobacter sp. and Pandoraea sp.,respectively.The optima l temperature, pH and phylogenetic relationship of bacterium strain PJ053 werestudied. The growth rate of PJ053 was slower tha n that of E coli. JM109. The 30℃and 7.0were the optima l temperature and pH for PJ053. The colony was smooth and glossy, regularity,round borderlin and bright yellow. The shape of strain was short-rhabditiform. Thephylogenetic analysis of 16S rDNA sequence showed that PJ053 formed a cluster with 4unclutured bacteria, but the genetic dista nce was far from Arthrobacter sp 14, etc.The genomiclibra ryof strain PJ053 was constructed and two positive clones JM109(pUCW402) and JM109 (pUCW331) were screened out for the expression of dioxygenase bythe ability to form yellowish ring-fission products. GenSCAN software and BLAST analysisshowed that a 2,3-dihydroxybiphenyl dioxygenase (23DHBD) gene of 933 bp and an ankyringene were found in the 3359 bp exogenous fragment of pUCW402. A hydroxylase ordehydrogenase gene could exist in the upstream of 23DHBD gene. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPCD) gene and a 5-carboxymethyl -2-hydroxymuconate semia ldehyde dehydrogenase were located in the inserting sequence ofpUCW331. Maybe a meta l dependent phosphohydrolase also existed in pUCW331.Thephylogeneticanalysisshowedthat23DHBDfromstrainPJ053formedadeepbra nchseparatefromaclustercontainingmostknown23DHBDinGenBank.Southernhybridizationconfirmed for the first time that the 23DHBD gene was existed in the genomicDNA ofArthrobacter sp. PJ053.In order to test the gene function, recombina nt bacteriaBL21 (pETW402) and BL21(pETW331) were constructedtoexpress23DHBD, HPCD and enzyme activity analysis .The 23DHBD and HPCD both were of the ability to express the dioxygnease to formyellowish ring-fission product 2-hydroxymuconic semia ldehyde on spraying colonies with 50mM catechol. Theexpressionlevel of 23DHBD and HPCD inBL21 (pETW402) and BL21(pETW331) were highercompared with therecombinantbacteriaJM109 (pUCW402),JM109 (pUCW331) andstrain PJ053. We observed that 23DHBD was not absolute specific.The enzyme activity was higher with 2,3-dihydroxybiphenyl as a substrate tha n with catechol.The substrate specificity assay suggested that 23DHBD was essentia l for cleava ge of bi-cyclicaromatic compounds during the course of aromatic compound biodegradation in Arthrobactersp. strain PJ053. The soluble fusion proteins 23DHBD and HPCD were purified after expend ingcultivation by Ni-NTA affinity chroma tography. Western hybrid ization of 23DHBD,HPCD from genetic engineered bacteria and strain PJ053 showed that the protein levelJM109 (pUCW402) and BL21(pETW402) were higher than that of strain PJ053. Thesame result was also obtained in HPCD.In this study, the primer was designed according to CarB gene of strain Sphingomonassp. KA1 and CarB (2'- aminobiphenyl- 2,3- diol dioxygenase gene) were amplified byPCR with genomic DNA of Sphingomonas sp. JS061 and Hydrogenophaga intermed iaJH062 as the template. Substrate coloration confirmed that the enzyme encoded by CarBwas of the ability to transform 2,3-dihydroxybiphenyl to 2-hydroxy -6-oxo -6-phenylhexa -2,4- dienoic acid . Sequence alignment showed the CarB sequences fromJS061 and JH062 were 100% identity with that of KA1 and GTIN11. The protein productof CarB was the same with that of CA10 and consisted of CarBa and CarBb whichmolcular weights were 29KD and 10KD,respectiely. The clone of CarB gene will provid evaluable information for studying the carbazole biodegradation gene cluster.