Molecular Characterization Of Catabolism Of 4-Nitrophenol In Pseudomonas Sp. WBC-3

Nitroaromatic compounds are common starting materials for the synthesis ofcomplex industrial N-containing compounds and can be major environmentalcontaminants, hazardous metabolic intermediates or dead-end products.Microorganisms play an important role in transforming these recalcitrantcontaminants and in the associated recycling of the nitrogen. In the pastdecades, the intensive research in this area has led to dramatic progress inunderstanding the microbial strategies and associated mechanisms for thedegradation of nitroaromatic compounds. A combination of increasingcommercial interest and advances in our understanding of the genetic andbiochemical basis of biodegradation is expected to produce a more rationalapproach to bioremediation technology.This dissertation is composed of three parts.The first part is determination of the molecular basis for 4-nitrophenoldegradation in Pseudomonas sp.strain WBC-3.WBC-3 can utilize methyl parathion (MP) and 4-nitrophenol (4-NP) as the solesource of carbon, nitrogen and energy. The key enzyme which catalyses theinitial reaction in MP degradation of WBC-3 is methyl parathion hydrolase(MPH), which converts MP to 4-NP stoichiometrically.To date, several 4-NP degrading bacteria have been isolated and theirdegradation pathways have been studied in details. The degradation pathway inwhich 4-NP is converted to maleylacetate via hydroquinone (hydroquinonepathway) was preferentially found in Gram-negative bacteria. The degradationpathway in which 4-NP is converted via 4-nitrocathechol (4-NCA) andhydroxyquinol (hydroxyquinol pathway) was preferentially found inGram-positive bacteria.The results of this study indicate that Pseudomonas sp. strain WBC-3 degraded4-NP through the typical hydroquinone pathway proposed by Spain inMoraxella (Spain et al, Appli.Environ.Microbiol., 1991: 812). The pathway proceeds through oxidative elimination of the nitro group to form benzoquinonewhich is subsequently reduced to hydroquinone in the presence of NADPH. Thelatter was cleavaged resulting in the formation ofγ-hydroxymuconicsemialdehyde by a soluble enzyme. Upon addition of catalytic amounts ofNAD~+,γ-hydroxymuconic semialdehyde was converted to maleylacetic acid,which enters the tricarboxylic acid cycle through theβ-ketoadipic acidpathway.Although many studies of 4-NP degradation have been reported, geneticinformation on 4-NP degradation remains limited and the only characterized4-NP degradation gene cluster originated from a Gram-positive bacterium,Rhodococcus opacus SAO101, which degradated 4-NP via hydroxyquinolpathway. No genes encoding degradation of 4-NP through hydroquinonepathway has been reported so far. Therefore, it was of great interest to cloneand characterize the catabolic genes involved in 4-NP metabolism to verify thehydroquinone pathway proposed by Spain. In particular, functionalcharacterization of the 4-nitrophenol monooxygenase, the enzyme involoved inthe initial reaction of the pathway, is highly desirable.In this report, a novel 4-NP degradation gene cluster from Gram-negativebacterium, Pseudomonas sp. WBC-3, was identified and characterized. First, abenzoquinone reductase gene, which is presumed to play a role in convertingbenzoquinone to hydroquinone in 4-NP degradation, was amplified by PCRbased on the conserved sequences. Second, the flanking regions of this genefragment were cloned and sequenced by genomic walking strategy. Finally, atotal of 14 kb contig was generated by assembling the sequence acquired fromthree genomic walking reactions. Fourteen possible ORFs were identified inthis contig and annotations of these ORFs were completed based on the resultsof BLAST analyses. Of these ORFs, the deduced amino acid sequence of PnpA,which was proposed to encode the para nitrophenol monooxygenase(PNPMO),shows moderate homology (25% identity) with pentachlorophenol4-monooxygenase of Sphingobium chlorophenolicum ATCC 39723 and pnpCappears to encode the dioxygenase for the cleavage of hydroquinone. Genesencoding benzoquinone reductase (pnpB),γ-hydroxymuconic semialdehydedehydrogenase (pnpD) and maleylacetic acid reductase (pnpE) were alsoidentified. Analyses of the sequenced region demonstrates that the genes involoved in thedegradation of 4-NP in WBC-3 were arranged in at least three operons, pnpBand pnpA are adjacent to each other but transcribed in opposite directions whilethe genes encoding the lower pathway of 4-NP degradation, pnpCDE, arenearby in operonic association with several open reading frames coding forproteins of unknown function. A putative regultor was also found upstream ofpnpCDE operon. Sequence alignment and phylogenetic anaylis indicate PnpArepresents a new member of FAD monooygenases and PnpC is a close relativeto hydroxyquinol 1, 2-dioxygenase family.In order to confirm the function of the putative genes, pnpA and pnpC wereexpressed in E.coli respectively. Conversion of 4-NP to hydroquinone wasobserved when expressed pnpA in E.coli with concomitant release of nitrite.Furthermore, either pnpA or pnpC deleted strain completely lost the ability togrow on 4-NP as sole carbon source. These results clearly suggested that bothpnpA and pnpC play essential roles in 4-NP mineralization in strain WBC-3.Characterization of PnpA as 4-nitrophenol monoxygenase was also conductedin the current study. PnpA demonstrates a narrow substrate range and E. colicells expressing pnpA can only attack 4-nitrophenol or 4-nitrocatechol. Theenzyme activity requires both FAD and NADPH as cofactors. NADH can serveas electron donor for the reaction but was less effective as NADPH.Interestingly, the reaction in crude extract required 2 mol of NADPH for theoxidation of each mol of 4-nitrophenol. These data suggest that the initialmonooxygenase converted 4-nitrophenol into benzoquinone at the expense of 1mol NADPH. The benzoquinone was then reduced to hydroquinone bybenzoquinone reductase at the expense of an additional mol of NADPH.The second part of this dissertation focuses on the chemotaxis features ofPseudomonas sp. strain WBC-3 toward aromatic compounds.The drop assay was used to show that multiple aromatic compounds arechemoattractants for Pseudomonas sp. strain WBC-3. Among thesechemoattractants, only 4-nitrophenol, 4-nitrocatechol and hydroquinone can bedegraded or transformed by WBC-3. The results presented here clearlysuggested that the chemotactic response of WBC-3 is constitutive and ismetabolism-independent based on the fact that (1) non-metabolizablecompounds are also attractants for WBC-3 and (2) the catabolic mutant strains do not affect its chemotaxis behavior. It was suggested that strain WBC-3 has aβ-ketoadipate chemotaxis system that responds to a wide range of aromaticcompounds, which is similar to that present in Pseudomonas PRS2000, themodel organism for chemotaxis study. The broad specificity of this chemotaxissystem works as advantage in WBC-3 and other soil bacterial because itsfunctions to dectect diverse carbon sources.The third part of this dissertation is the annotation of DNA sequence from thecatabolic plasmid pZWL0 of Pseudomonas sp. WBC-3.pZWL0 carrys the catabolic gene mph (methyl parathion hydrolase), which isresponsible for the conversion of methyl parathion to 4-nitrophenol inPseudomonas sp WBC-3. Analysis of the partial complete nucleotide sequence(76 kb) revealed 62 potential opening reading frames (ORFs) and most of thoseare related to the core functions of plasmid such as replication, stablemaintenance and transfer. The plasmid maybe attribute to the IncPincompatibility group based on the identities of the putative replicon protein ofpZWL0. However, the sequence and organization of the backbone genes ofpZWL0 show lower identities with known plasmids, which may imply itsdistant relationships with other catabolic plasmids.The sequence analysis also reveal that the mph gene in WBC-3 is flanked bytwo copies of insertion sequence (IS6100), which is the typical character ofclassⅠtransposon. This transposon, designated Tn-mph1, is a composition of alarge transposon, designated Tn-mph2, which is a classⅡtransposon accordingto its sequence and organization characters.To data, two types of organophosphate hydrolase had been identified, oneis MPH-like and the other is OPD-like. Genes encoding both enzymes areproposed to be physically located in a typical transposon, which may beresponsible for the widely distribution of these enzymes. However, these twokinds of enzymes share poor sequence identities and phylogenetic analysissuggested that organophosphate hydrolases were multioriginate and MPH,which evolved independently from OPD, diverged fromβ-lactamase family.