Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.     

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

Summary A bioconversion process was demonstrated by manipulation of catabolic genes. Catabolic intermediates of various biphenyl compounds could be efficiently produced by Pseudomonas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydrogenase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.     

A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6

A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.     

Polychlorinated biphenyl/biphenyl degrading gene clusters in Rhodococcus sp. K37, HA99, and TA431 are different from well-known bph gene clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Phylogeny and a new species of Sparassis (Polyporales, Basidiomycota): evidence from mitochondrial atp6, nuclear rDNA and rpb2 genes

Three nuclear genes, lsu-rDNA (encoding nuclear large subunit rDNA), ITS (encoding the rDNA internal transcribed spacers and 5.8 S rDNA) and rpb2 (encoding the second largest subunit of RNA polymerase II), and the mitochondrial gene atp6 (encoding the sixth subunit of ATP synthase), were sequenced from all recognized Sparassis lineages. Sparassis latifolia sp. nov. from boreal coniferous forests in China is described based on morphological, ecological, geographical and molecular data. The nuclear gene phylogeny strongly supported groups corresponding to morphological differences, geographic distribution and host shifts among species that produce clamp connections, such as S. crispa from Europe, S. radicata from western North America and S. latifolia from Asia. The atp6 phylogeny however showed no divergence among these three species. For clampless Sparassis species, such as S. spathulata from eastern North America, S. brevipes and a new species from Europe, the atp6 phylogeny was congruent with the nuclear gene phylogeny. Sparassis cystidiosa is basal in the nuclear tree but sister to S. brevipes-S. spathulata clade in the ATP6 tree. The differences between the phylogenetic inferences from the atp6 gene and those from nuclear genes within Sparassis species are discussed.     

Multiplicity of 2,3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37

Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.     

Plasmids for biphenyl, chlorobiphenyl and metatoluylate degradation from Pseudomonas putida

Pseudomonas putida strain SU83, harbors the pBS311 plasmid coding for the degradation of biphenyl, 2- and 4-chlorbiphenyl, meta- and paratoluylate. The insertional mutants of the plasmid obtained by the transposon Tn5 insertion were isolated. One of the mutants was used for cloning of the biphenyl degradation genes. The plasmid pBS311:: Tn5 DNA was inserted into the BamHI site of the plasmid pBR322 and cloned. 11 recombinants of 354 tested were treated with 0.1% solution of 2,3-dioxybiphenyl. One of them has acquired the yellow colour testifying to conversion of 2,3-dioxyphenyl to "2-hydroxy-6-keto-6-phenylhexa-2,4-diene acid. The recombinant plasmid pBS312 from this clone is 10.5 kb in size, the size of the insert being 6.2 kb. Escherichia coli SU185 cells harbouring pBS312 are able to support metacleavage of 2,3-dioxybiphenyl, 3-methylcatechol and catechol, but not of 4-methylcatechol. The results suggest the cloned fragment to contain a gene for 2,3-dioxybiphenyl-1,2-dioxygenase, the third enzyme for biphenyl catabolism.     

Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation.

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.     

Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli

Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.     

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance,resistance gene identification,and heterologous expression

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dα6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dα6, providing convincing evidence that Dα6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dα6 alone, co-expression of Dα6 with an additional nAChR subunit, Dα5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.     

A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.     

Cloning,expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1,a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers.The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene.In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves.The peak expression signal was observed in mesophyll cells under low phosphorus(P)induction.A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris.At the meantime,the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters de- ficient.Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.     

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.     

Cloning and analysis of the pseudogene for human epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT)

1. This gene completely lacks the intervening sequences. 2. This gene is truncated at the 5' end peptide encoding region by 433 base pairs (bp). 3. The 502 bp of this gene containing poly(A) signal are completely identical to the 3' half of mRNA encoding region of functional gene. 4. This gene has a poly(A) tail and is flanked by direct repeat of 6 bp. 5. Here we report for the first time the complete sequence of a human pseudogene for phenylethanolamine N-methyltransferase and this is the first report of cloning of pseudogene for catecholamine biosynthetic enzymes.     

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.