Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

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.     

Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

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.     

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27–33% and 32–38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCbphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.     

Complete Genome Sequence of Acidovorax sp. Strain KKS102, a Polychlorinated-Biphenyl Degrader

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.     

Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102.

Two genes involved in the degradation of biphenyl were isolated from a gene library of a polychlorinated biphenyl-degrading soil bacterium, Pseudomonas sp. strain KKS102, by using a broad-host-range cosmid vector, pKS13. When a 3.2-kilobase (kb) PstI fragment of a 29-kb cosmid DNA insert was subcloned into pUC18 at the PstI site downstream of the lacZ promoter, Escherichia coli cells carrying this recombinant plasmid expressed 2,3-dihydroxybiphenyl dioxygenase activity. Nucleotide sequencing of the 3.2-kb PstI fragment revealed that there were two open reading frames (ORFI [882 base pairs] and ORFII [834 base pairs], in this gene order). Results of analysis of Tn5 insertion mutants and unidirectional deletion mutants suggested that the ORFI coded for 2,3-dihydroxybiphenyl dioxygenase. When the sequence of ORFI was compared with that of bphC of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa, N. Arima, and T. Miyazaki, J. Bacteriol. 169:427-429, 1987), the homology was 68%, with both strains having the same Shine-Dalgarno sequence. The result of gas chromatography-mass spectrometry analysis of the metabolic product suggested that the ORFII had meta cleavage compound hydrolase activity to produce benzoic acid. DNA sequencing suggested that these two genes were contained in one operon.     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.     

Growth of an estuarinePseudomonas sp. on polychlorinated biphenyl

In the course of a study of the microbial degradation of polychlorinated biphenyls (PCB), 16 cultures, representing 5 genera of bacteria, were isolated. APseudomonas sp. was found to degrade mixtures of PCB (Aroclor 1254) and pure isomers, i.e., hexachlorobiphenyl (HCB). Degradation was found to be variable, depending on length of incubation, purity, and degree of chlorination of the biphenyl molecule. In addition to apparent degradation of PCB, it was observed that PCB stimulated growth and oxygen uptake. Bioaccumulation of PCB residues was suggested since¹⁴-Clabeled material derived from¹⁴C-PCB accumulated on or in the bacterial cells. The potential degradation and utilization of PCB by microorganisms suggests that this may be an important mechanism for removal or mobilization of PCB into food chains in the estuarine environment.     

Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400.     

Characterization of two biphenyl dioxygenases for biphenyl/PCB degradation in A PCB degrader, Rhodococcus sp. strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.     

Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

Conjugation, a major type of horizontal gene transfer in bacteria, involves transfer of DNA from a donor to a recipient using donor‐encoded conjugation machinery. Using a high‐throughput screen (Tn‐seq), we identified genes in recipients that contribute to acquisition of the integrative and conjugative element ICEBs1 by Bacillus subtilis. We found that null mutations in some genes caused an increase, and others a decrease in conjugation efficiency. Some mutations affected conjugation only when present in recipients. Other mutations affected conjugation when present in donors or recipients. Most of the genes identified are known or predicted to affect the cell envelope. Several encode enzymes involved in phospholipid biosynthesis and one encodes a homologue of penicillin‐binding proteins. Two of the genes identified also affected conjugation of Tn916, indicating that their roles in conjugation may be general. We did not identify any genes in recipients that were essential for ICEBs1 conjugation, indicating that if there are such genes, then these are either essential for cell growth or redundant. Our results indicate that acquisition of ICEBs1, and perhaps other conjugative elements, is robust and not easily avoided by mutation and that several membrane‐related functions affect the efficiency of conjugation.     

Structural alteration of linear plasmids encoding the genes for polychlorinated biphenyl degradation in Rhodococcus strain RHA1

Polychlorinated biphenyl (PCB) tolerant derivatives of a strong PCB degrader, Rhodococcus strain RHA1, were selected after growth in the presence of 100 g/ml PCBs. Some of the derivatives did not grow on biphenyl but accumulated a yellow coloured metabolite suggesting a defect in the meta-ring-cleavage compound hydrolase step encoded by the bphD gene. Other derivatives failed to grow on biphenyl and exhibited little PCB transformation activity suggesting a defect in the initial ring-hydroxylation dioxygenase step encoded by the bphA gene. These organisms had a structural alteration in the linear plasmids coding for the bph genes in RHA1, which included the bph gene deletion. When a bphD containing plasmid was introduced into a tolerant derivative, RCD1, which was shown to have a bphD deletion, the defect in the growth on biphenyl of RCD1 was overcome. The bph gene deletion seems to play a key role in these tolerant derivatives thereby suggesting that the toxic metabolic intermediate would be a main cause of the growth inhibition of RHA1 in the presence of high concentration PCBs.     

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida.

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)