[Show abstract][Hide abstract] ABSTRACT: In plants, serine residues in extensin, a cell wall protein, are glycosylated with O-linked galactose. However, the enzyme that is involved in the galactosylation of serine had not yet been identified. To identify
the peptidyl serine O-α-galactosyltransferase (SGT), we chose Chlamydomonas reinhardtii as a model. We established an assay system for SGT activity using C. reinhardtii and Arabidopsis thaliana cell extracts. SGT protein was partially purified from cell extracts of C. reinhardtii and analyzed by tandem mass spectrometry to determine its amino acid sequence. The sequence matched the open reading frame
XP_001696927 in the C. reinhardtii proteome database, and a corresponding DNA fragment encoding 748 amino acids (BAL63043) was cloned from a C. reinhardtii cDNA library. The 748-amino acid protein (CrSGT1) was produced using a yeast expression system, and the SGT activity was
examined. Hydroxylation of proline residues adjacent to a serine in acceptor peptides was required for SGT activity. Genes
for proteins containing conserved domains were found in various plant genomes, including A. thaliana and Nicotiana tabacum. The AtSGT1 and NtSGT1 proteins also showed SGT activity when expressed in yeast. In addition, knock-out lines of AtSGT1 and knockdown lines of NtSGT1 showed no or reduced SGT activity. The SGT1 sequence, which contains a conserved DXD motif and a C-terminal membrane spanning region, is the first example of a glycosyltransferase with type I membrane protein
topology, and it showed no homology with known glycosyltransferases, indicating that SGT1 belongs to a novel glycosyltransferase gene family existing only in the plant kingdom.
[Show abstract][Hide abstract] ABSTRACT: NtP4H1.1 is a Golgi-localizing type II integral membrane protein. Mutations in the cytoplasmic tail direct the protein to the endoplasmic reticulum (ER). We expressed a GFP fusion protein containing the mutant tail and the transmembrane region in tobacco BY-2 cells, and found that the protein localized in the Golgi. Therefore NtP4H1.1 contains multiple targeting information in different regions.
[Show abstract][Hide abstract] ABSTRACT: A microbial consortium that reductively dechlorinates trichloroethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene with methanogenesis was enriched from chloroethene-contaminated soil from Japan. Dechlorination activity was maintained for over 4 years. Using quantitative polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis targeting the "Dehalococcoides" 16S rRNA gene, four strains were detected. Their growth and dechlorination activities were classified into two types: one that grows by converting cis-DCE to ethene and the other that grows by converting cis-DCE to VC. Then, the vcrA and bvcA genes encoding cis-DCE/VC reductive dehalogenases were detected. Inhibitors of methanogenesis (2-bromoethanesulfonate) and sulfidogenesis (molybdate) led to accumulation of cis-DCE and of VC respectively. These results suggest that methanogens and sulfate-reducing bacteria can play a significant role in dechlorination by "Dehalococcoides."
[Show abstract][Hide abstract] ABSTRACT: Plant specific O-glycosylation of proteins includes the attachment of arabinogalactan to hydroxyproline (Hyp) residues. These Hyp residues are generated from peptidyl proline residues by the action of prolyl 4-hydroxylase which requires the ferrous ion. We investigated the effect of the ferrous chelator, 2,2'-dipyridyl on tobacco plants, and found that such treatment reduced the arabinogalactosylation of proteins.
[Show abstract][Hide abstract] ABSTRACT: Desulfitobacterium sp. strain Y51 exhibits a strong dechlorinating activity for tetrachloroethene (PCE), converting it to cis-1,2-dichloroethene via trichloroethene by the action of the PceA reductive dehalogenase (encoded by pceA). The gene organization around the pceA gene cluster was determined to be in the following order: orf4, orf3, ISDesp1, pceA-B-C-T-mcpA, and ISDesp2, where the pceA gene cluster is surrounded by two nearly identical copies of the ISDesp insertion sequence. Serial subculture of strain Y51 gave rise to variants that abolished the PCE-dechlorination activity. Southern hybridization analysis revealed two types of variants termed small deletion (SD) and large deletion (LD). The characterization of both variants revealed a genetic rearrangement around the pceAB gene cluster. In variant SD, ISDesp1 comprised of 1,572 bp was deleted, which includes the tnpAa encoding IS256 family transposase and unknown orf1. The ISDesp1 contained the inverted terminal repeat sequence and a -35 promoter stretch just upstream of the pceA gene, indicating that this IS element is involved in the formation of the variant SD. Loss of the pceA transcription changed the variant SD to the PCE-nondechlorinating phenotype. The variant LD lost the 6.5-kb region, including one copy of ISDesp and the pceABCT-mcpA gene cluster, confirming that the homologous recombination is associated with the emergence of this variant.
[Show abstract][Hide abstract] ABSTRACT: A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 microM and as low as 0.06 microM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min(-1) mg protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.
Journal of Industrial Microbiology and Biotechnology 01/2006; 32(11-12):534-41. DOI:10.1007/s10295-005-0252-z · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol x min(-1) x mg of protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.
Journal of Bacteriology 08/2002; 184(13):3419-25. DOI:10.1128/JB.184.13.3419-3425.2002 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FD(BphA3)), and a ferredoxin reductase (FDR(BphA4)). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP, FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISP(TodC1BphA2), FD(BphA3), and FDR(BphA4) exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP(TodC1BphA2) and ISP(BphA1A2) were present as heterohexamers, whereas ISP(TodC1C2) was present as a heterotetramer. The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.
[Show abstract][Hide abstract] ABSTRACT: A strict anaerobic bacterium, strain Y51, was isolated from soil contaminated with tetrachloroethene (PCE). Strain Y51 is capable of very efficiently dehalogenating PCE via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at concentrations as high as 960 microM and as low as 0.6 microM. Strain Y51 was gram-negative, motile with some lateral flagella, and curved rod-shaped. On the basis of the 16S rDNA sequence, the organism was identified to be a species within the genus Desulfitobacterium. Strain Y51 also had dehalogenation activities toward polychloroethanes such as hexa-, penta-, and tetrachloroethanes, from which dichloroethenes were produced as the final products. The cell extracts mediated the dehalogenation of PCE with reduced methyl viologen as an electron carrier at the specific rate of 5.0 nmol min(-1) mg cell protein(-1) (pH 7.2, 37 degrees C). Dehalogenation was highly susceptible to air oxidation, and to potential alternative electron acceptors such as nitrite or sulfite.
[Show abstract][Hide abstract] ABSTRACT: A laboratory test was conducted to examine the combined effect of bioaugmentation of an anaerobic bacterial Desulfitobacterium sp. strain Y-51 and addition of zero-valent iron (Fe0) on the reductive dechlorination of tetrachloroethylene (PCE) in a non-sterile soil slurry. Introduction of a strain Y-51 culture in soil (3 mg vss (volatile suspended solids)/kg soil) containing PCE (at 60 μmol/kg soil) led to complete conversion of PCE to cis-1,2-dichloroethylene (cis-DCE) within 40 d. Treatments of the same soil slurry with Fe0 (0.1–1.0%) resulted in extended PCE dechlorination to ethylene (ETH) and ethane (ETA). The combined use of a strain Y-51 culture and Fe0 showed effective dechlorination of PCE than did the individual use. The cis-DCE produced from biological PCE dechlorination by strain Y-51 was totally converted to non-chlorinated end products by the following chemical reduction by Fe0. Furthermore, anaerobic corrosion of Fe0 was found to stimulate the biological reductive dechlorination of PCE by keeping proper levels of pH and oxidation-reduction potential (ORP) and by producing cathodic hydrogen, which might be used as an electron donor for respiratory PCE dechlorination. These findings suggest that the combined use of bacterial strain Y-51 and Fe0 is effective for practical treatment of PCE and other chlorinated ethylenes in contaminated sites.
Journal of Bioscience and Bioengineering 02/2001; 92(5-92):453-458. DOI:10.1016/S1389-1723(01)80295-4 · 1.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We constructed hybrid Pseudomonas strains in which the bphA1 gene (coding for a large subunit of biphenyl dioxygenase) is replaced with the todC1 gene (coding for a large subunit of toluene dioxygenase of Pseudomonas putida Fl) within chromosomal biphenyl-catabolic bph gene clusters. Such hybrid strains gained the novel capability to grow on a wide range of aromatic hydrocarbons, and, more interestingly, they degraded chloroethenes such as trichloroethylene and cis-1,2-dichloroethylene very efficiently.
Journal of Bacteriology 08/1996; 178(14):4039-46. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.
[Show abstract][Hide abstract] ABSTRACT: Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 [Taira et al., J. Biol. Chem. 267 (1992) 4844-4858] and toluene Dox encoded by the gene cluster, todC1C2BA in P. putida strain F1 [Zylstra et al., J. Biol. Chem. 264 (1989) 14940-14946], show high homologies (approx. 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities. We constructed hybrid gene clusters by replacing the gene component(s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox. Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodC1::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4). Moreover, hybrid Dox (composed of TodC1::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodC1C2BA). On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate. These results suggest that (i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and (ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes.
[Show abstract][Hide abstract] ABSTRACT: bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.
Journal of Bacteriology 09/1993; 175(16):5224-32. · 2.69 Impact Factor