Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.
ABSTRACT: Pseudomonas sp. strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway. We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 [ORF1]) A4 and found four ORFs in this region. As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp. strain CF600 (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively. The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), whose function is unknown. The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes. The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively. The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4. The functions of ORF1 and ORF4 are unknown. The features of this bph gene cluster are discussed.
Project description:The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida F1 and have been named bphA, bphE, bphF, and bphG. From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase. Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a [2Fe-2S] cluster. Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding. Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified. Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.
Project description:The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.
Project description:We report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.
Project description:A polychlorinated biphenyl (PCB)/biphenyl degradation gene cluster in Acidovorax sp. strain KKS102, which is very similar to that in Tn4371 from Cupriavidus oxalaticus A5, was transferred to several proteobacterial strains by conjugation. The mobilized DNA fragment consisted of 61,807 bp and carried genes for mating-pair formation (mpf), DNA transfer (dtr), integrase (int), and replication-partition proteins (rep-parAB). In the transconjugants, transferred DNA was integrated at ATTGCATCAG or similar sequences. The circular-form integrative and conjugative element (ICE) was detected by PCR, and quantitative PCR analyses revealed that, in KKS102 cells, the ratio of the circular form to the integrated form was very low (approximately 10(-5)). The circular form was not detected in a mutant of the int gene, which was located at the extreme left and transcribed in the inward direction, and the level of int transcriptional activity was much higher in the circular form than in the integrated form. These findings clearly demonstrated that the genes for PCB/biphenyl degradation in KKS102 cells are located on an ICE, which was named ICE(KKS102)4677. Comparisons of similar ICE-like elements collected from the public database suggested that those of beta- and gammaproteobacteria were distinguishable from other ICE-like elements, including those in alphaproteobacteria, with respect to the gene composition and gene organization.
Project description:Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Project description:We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.
Project description:Pseudomonas putida KF715 (NBRC 110667) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report a complete genome sequence of the KF715 strain, which comprises a circular chromosome and four plasmids. Biphenyl catabolic genes were located on the largest plasmid, pKF715A.
Project description:A gram-positive polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, metabolizes biphenyl through the 2-hydroxypenta-2,4-dienoate (HPD) and benzoate metabolic pathways. The HPD metabolic pathway genes, the HPD hydratase (bphE1), 4-hydroxy-2-oxovalerate aldolase (bphF1), and acetaldehyde dehydrogenase (acylating) (bphG) genes, were cloned from RHA1. The deduced amino acid sequences of bphGF1E1 have 30 to 58% identity with those of the HPD metabolic pathway genes of gram-negative bacteria. The order of these genes, bphG-bphF1-bphE1, differs from that of the HPD metabolic pathway genes, bphE-bphG-bphF, in gram-negative degraders of PCB, phenol, and toluene. Reverse transcription-PCR experiments indicated that the bphGF1E1 genes are inducibly cotranscribed in cells grown on biphenyl and ethylbenzene. Primer extension analysis revealed that the transcriptional initiation site exists within the bphR gene located adjacent to and upstream of bphG, which is deduced to code a transcriptional regulator. The respective enzyme activities of bphGF1E1 gene products were detected in Rhodococcus erythropolis IAM1399 carrying a bphGF1E1 plasmid. The insertional inactivation of the bphE1, bphF1, and bphG genes resulted in the loss of the corresponding enzyme activities and diminished growth on both biphenyl and ethylbenzene. Severe growth interference was observed during growth on biphenyl. The growth defects were partially restored by the introduction of plasmids containing the respective intact genes. These results indicated that the cloned bphGF1E1 genes are not only responsible for the primary metabolism of HPD during growth on both biphenyl and ethylbenzene but are also involved in preventing the accumulation of unexpected toxic metabolites, which interfere with the growth of RHA1.
Project description:Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.
Project description:Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.