Pseudomonas putida HS12 isolated from soil was able to use nitrobenzene (NB) as a sole source of carbon, nitrogen, and energy and was found to possess a partial reductive pathway for the degradation of NB. From LC/MS and $^1H-NMR$ spectroscopic analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-chloronitrobenzenes (3- and 4-CNBs) to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For complete mineralization of CNBs, Rhodococcus sp. HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a co-culture. This co-culture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. The degradation pathways for NB, 3-CNB, and 4-CNB by the two isolated strains were also proposed.
Pseudomonas putida HS12 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 the two plasmids, pNB1 and pNB1. 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 E. 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 pNB1, 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 of 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 which seems to be encoded on pNB2.
The amiophenol meta cleavage operon of Pseudomonas putida HS12 encodes a set of enzymes which transform 2-AP (2-aminophenol) to TCA cycle intermediates, pyruvate and acetyl CoA via extra diol cleavage of 2-AP. The genetic organization of the AP operon was characterized by cloning of the meta-cleavage genes into an expression vector and identified their products in E. coli by functional analysis. Functional analysis, as well as comparison study of amino acid sequence deduced from whole DNA sequence, showed that the AP cleavage operon contains 11 genes, nbzR, nbzCaCbDGFEIH, and unidentified 2 ORFs. The nbzR gene encodes putative transcriptional regulatory protein. The nbzCaCbDGFEIH genes encode β- and α-subunits of 2-AP 1,6-dioxygenase, AMS (2-aminomuconic semialdehyde) dehydrogenase, OP (2-oxopent-4-enoate) hydratase, OC (4-oxalocrotonate) decarboxylase, AM (2-aminomuconate) deaminase, ACT (acetaldehyde) dehydrogenase, and HOV (4-hydroxy-2-oxovalerate) aldolase, respectively. From comparison study of deduced amino acid of each enzyme and genetic organization of AP catabolic operon with other meta cleavage operon including TOL, NAH, and DMP operon, it is concluded that AP catabolic operon has divergently evolved from ancestral meta cleavage operon.
대표적인 독성 방향족화합물로 알려진 nitrobenzene (NB)와 chloronitrobenzenes (CNBs)를 분해하는 미생물은 탐색하고 분해기작을 밝혔다. 먼저 NB를 분해하는 미생물을 분리하였는데, 이 균주는 Pseudomonas putida HS12로서 NB이외에 CNBs를 분해하는 활성을 갖고 있었다. LC/MS와 NMR 분석을 통해서 P. putida HS12가 NB과 CNBs를 분해하면서 생성하는 중간산물들의 구조를 확인함으로서 이들 니트로 방향족화합물들의 대사경로를 규명하였다. 또한, 염소방향족화합물을 분해하는 Rhodococcus sp. HS51을 분리하였는데, 이 두 균주의 혼합배양에서 NB와 CNBs가 partial reductive pathway, acetylation, ring cleavage pathway의 경로를 통해서 분해됨을 확인하였다.
NB를 분해하는 HS12는 두 개의 plasmid를 갖고 있는데, 이들 plasmid에 NB의 모든 catabolic gene이 있음을 확인하여 대장균에 cloning 하고 각 subclone들의 functional analysis를 하였다. 그 결과 pNB2 (43.8-kb)에 NbzB (mutase)의 gene이 있었으며, pNB1 (59.1-kb)에 NbzA (nitroreductase)와 NbzCDE (dioxygenase, dehydrogenase, deaminase) 두 개의 functional unit가 있음을 확인하였다. 특히, AP 의 meta ring cleavage pathway는 operon의 형태로 발견되었는데, 이 AP catabolic operon이 있는 11-kb fragment를 DNA sequencing하고 여기에서 알 수 있는 ORF들의 amino acid residue를 지금까지 알려진 다른 meta cleavage operon과 비교하였으며, 각 subclone들의 functional analysis로부터, 각 ORF의 function을 밝혔다. AP catabolic operon은 1개의 regulatory gene인 nbzR, 8개의 structural gene인 nbzCaCbDGFEIH, 그리고 2개의 unidentified gene으로 구성되어 있었다. AP catabolic operon과 다른 meta cleavage operon의 비교 연구를 통해서 AP operon은 원시 meta operon으로부터 divergent evolution 하였는데, 그 분화시기는 다른 meta operon에 hydrolytic branch가 삽입되기 전에 일어났을 것으로 추정된다. 그리고, 이 AP operon은 원시 meta operon으로부터 AP에 specific한 APDO, AMSDH, 그리고 DA 효소들을 진화과정중에 갖게 됨으로서 catechol이 아닌AP을 분해할 수 있게 된 것으로 보인다.