Identification and Characterization of the Nitrobenzene Catabolic Plasmids pNB1 and pNB2 in Pseudomonas putida HS12

Abstract

Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in Escherichia coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes nbzC, nbzD, and nbzE, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb SnaBI-SmaI fragment of pNB1 and clustered in the order nbzC-nbzD-nbzE as an operon. The nbzCDE genes, which are transcribed in the opposite direction of the nbzA gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.Release of massive amounts of nitroaromatic compounds into the environment has enabled a number of microorganisms to evolve the ability to mineralize these xenobiotic compounds. Nitrobenzene (NB), one of the recalcitrant nitroaromatic compounds, has been reported to be mineralized by microorganisms through oxidative (25) and partial reductive pathways (11,24). In addition, construction of a hybrid pathway for the degradation of NB was attempted with Pseudomonas putida TB103 (13). Recently, Pseudomonas pseudoalcaligenes JS45 was reported to have a partial reductive pathway, and several enzymes involved in NB catabolism were purified and characterized (10,12,19,37). We previously isolated P. putida HS12, which is able to utilize NB, and showed that P. putida HS12 possesses a partial reductive pathway (29).In the partial reductive pathway (Fig. 1), NB is first reduced to hydroxylaminobenzene (HAB) by the NB nitroreductase and then HAB is rearranged to 2-aminophenol (2-AP) by HAB mutase. 2-AP undergoes a meta ring cleavage to 2-aminomuconic 6-semialdehyde (2-AMS) by 2-AP 1,6-dioxygenase. 2-AMS dehydrogenase converts the resulting 2-AMS to 2-aminomuconate (2-AM), which is, by the action of 2-AM deaminase, finally deaminated to yield 4-oxalocrotonate, a wellknown intermediate in the catechol meta cleavage pathway. Some of the NB catabolic enzymes, such as NB nitroreductase (37), 2-AP 1,6-dioxigenase (19), 2-AMS dehydrogenase (12), and 2-AM deaminase (10) in P...

Conserved YjgF Protein Family Deaminates Reactive Enamine/Imine Intermediates of Pyridoxal 5′-Phosphate (PLP)-dependent Enzyme Reactions

“…Consistent with the activity demonstrated here, some enzymes that participate in other enamine deamination reactions can be defined as low identity members of the YjgF family (25-30% identical to S. enterica). In Pseudomonas pseudoalcaligenes, a YjgF-like protein was shown to deaminate the short-lived enamine 2-aminomuconate to 4-oxalocrotonic acid during the degradation of nitrobenzene (34), and the same reaction has been reported for homologs NbzE in nitrobenzene-degrading Pseudomonas putida HS12 (35) and AmnD in Pseudomonas sp. AP-3 (36).…”

Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process

“…Cells of P. putida strains were grown in defined mineral medium at 30°C as described elsewhere (37). Cells of Escherichia coli strains were cultured in Luria-Bertani medium at 37°C.…”

“…DNA manipulations, such as subcloning and transformation, were performed in accordance with the standard procedure described by Sambrook et al (41). Plasmids of P. putida HS12 were isolated as previously described (37). DNA fragments from pNB1 and pNB2 were cloned into pBluescript SK/KS and pUCP22, a broad-host-range vector for gram-negative bacteria.…”

“…However, despite intensive studies on the catabolic pathway of NB and characterization of the relevant enzymes, a detailed molecular basis of and a regulatory mechanism for NB catabolism remain yet to be elucidated. Recently, we reported the novel genetic organization of the NB catabolic gene clusters that are on the catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12 (37). All the genes except for that of mutase, which was found on pNB2, were clustered on pNB1.…”

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…strain AP-3 (191) and Pseudomonas sp. strain HS12 (147,148) use similar pathways and enzymes for nitrobenzene degradation to those of JS45. However, in AP-3, 2-aminomuconate may undergo decarboxylation prior to deamination during the formation of 2-oxo-4-pentenoate (190,191).…”