Title: Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor Miyazaki S, Tomita K, Yamane H, Kobayashi M, Asami T, Nakajima M Ref: Bioorganic & Medicinal Chemistry Lett, 28:2465, 2018 : PubMed
Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.
Plasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazole-degradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistance-nodulation-cell division family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared with pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.
Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.
BACKGROUND: Plasmids are extrachromosomal elements that replicate autonomously, and many can be transmitted between bacterial cells through conjugation. Although the transcription pattern of genes on a plasmid can be altered by a change in host background, the expression range of plasmid genes that will result in phenotypic variation has not been quantitatively investigated. RESULTS: Using a microarray with evenly tiled probes at a density of 9 bp, we mapped and quantified the transcripts of the carbazole catabolic plasmid pCAR1 in its original host Pseudomonas resinovorans CA10 and the transconjugant P. putida KT2440(pCAR1) during growth on either carbazole or succinate as the sole carbon source. We identified the operons in pCAR1, which consisted of nearly identical transcription units despite the difference in host background during growth on the same carbon source. In accordance with previous studies, the catabolic operons for carbazole degradation were upregulated during growth on carbazole in both hosts. However, our tiling array results also showed that several operons flanking the transfer gene cluster were transcribed at significantly higher levels in the transconjugant than in the original host. The number of transcripts and the positions of the transcription start sites agreed with our quantitative RT-PCR and primer extension results. CONCLUSION: Our tiling array results indicate that the levels of transcription for the operons on a plasmid can vary by host background. High-resolution mapping using an unbiased tiling array is a valuable tool for the simultaneous identification and quantification of prokaryotic transcriptomes including polycistronic operons and non-coding RNAs.
Title: Carbazole-degradative IncP-7 plasmid pCAR1.2 is structurally unstable in Pseudomonas fluorescens Pf0-1, which accumulates catechol, the intermediate of the carbazole degradation pathway Takahashi Y, Shintani M, Li L, Yamane H, Nojiri H Ref: Applied Environmental Microbiology, 75:3920, 2009 : PubMed
We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazole-degradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole- and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazole-degradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.
Title: The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids Takahashi Y, Shintani M, Yamane H, Nojiri H Ref: Biosci Biotechnol Biochem, 73:744, 2009 : PubMed
pCAR1 and pCAR2 are IncP-7 self-transmissible carbazole degradative plasmids. Their respective hosts showed clearly different conjugative host ranges. Their complete nucleotide sequences were virtually the same, and can be regarded as structurally the same plasmid, indicating that the difference in the conjugative host range was caused by host cell backgrounds.
We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.
Title: Characterization of novel carbazole catabolism genes from gram-positive carbazole degrader Nocardioides aromaticivorans IC177 Inoue K, Habe H, Yamane H, Nojiri H Ref: Applied Environmental Microbiology, 72:3321, 2006 : PubMed
Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.
The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.
Title: A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products Fushinobu S, Jun SY, Hidaka M, Nojiri H, Yamane H, Shoun H, Omori T, Wakagi T Ref: Biosci Biotechnol Biochem, 69:491, 2005 : PubMed
Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k(cat) values of the corresponding substrates. The Ile139 and Trp143 residues on helix alpha4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.
Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the beta-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding beta-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes gamma-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited beta-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important beta-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.
Title: Diversity of carbazole-degrading bacteria having the car gene cluster: isolation of a novel gram-positive carbazole-degrading bacterium Inoue K, Habe H, Yamane H, Omori T, Nojiri H Ref: FEMS Microbiology Letters, 245:145, 2005 : PubMed
Twenty-seven carbazole-utilizing bacterial strains were isolated from environmental samples, and were classified into 14 groups by amplified ribosomal DNA restriction analysis. Southern hybridization analyses showed that 3 and 17 isolates possessed the car gene homologs of Pseudomonas resinovorans CA10 and Sphingomonas sp. strain KA1, respectively. Of the 17 isolates, 2 isolates also have the homolog of the carAa gene of Sphingomonas sp. strain CB3. While the genome of one isolate, a Gram-positive Nocardioides sp. strain IC177, showed no hybridization to any car gene probes, PCR and sequence analyses indicated that strain IC177 had tandemly linked carAa and carC gene homologs whose deduced amino acid sequences showed 51% and 36% identities with those of strain KA1.
Title: Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains Shintani M, Habe H, Tsuda M, Omori T, Yamane H, Nojiri H Ref: Biotechnol Lett, 27:1847, 2005 : PubMed
The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.
Title: Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63 Takagi T, Habe H, Yoshida T, Yamane H, Omori T, Nojiri H Ref: Applied Microbiology & Biotechnology, 68:336, 2005 : PubMed
Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2',3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.
Genes involved in the degradation of fluorene to phthalate were characterized in the fluorene degrader Terrabacter sp. strain DBF63. The initial attack on both fluorene and 9-fluorenone was catalyzed by DbfA to yield 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone, respectively. The FlnB protein exhibited activities against both 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone to produce 9-fluorenone and 2'-carboxy-2,3-dihydroxybiphenyl, respectively. FlnD is a heteromeric protein encoded by flnD1 and ORF16, being a member of the class III two-subunit extradiol dioxygenase. FlnE was identified as a serine hydrolase for the meta-cleavage products that yield phthalate.
Several types of jasomonic acid (JA) derivatives, including JA--amino acid conjugates, a JA--biotin conjugate, a JA--dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA--binding proteins. These JA derivatives, excepting the JA--fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA--binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.
2-Hydroxy-6-oxo-6-(2(')-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system. The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans strain CA10 (ht-CarC(CA10)) and Janthinobacterium sp. strain J3 (ht-CarC(J3)) exhibited hydrolase activity toward 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate as the purified native CarC(CA10) did. ht-CarC(J3) was crystallized in the space group I422 with cell dimensions of a=b=130.3A, c=84.5A in the hexagonal setting, and the crystal structure of ht-CarC(J3) was determined at 1.86A resolution. The final refined model of ht-CarC(J3) yields an R-factor of 21.6%, although the electron-density corresponding to Ile146 to Asn155 was ambiguous in the final model. We compared the known structures of BphD from Rhodococcus sp. strain RHA1 and CumD from Pseudomonas fluorescens strain IP01. The backbone conformation of ht-CarC(J3) was better superimposed with CumD than with BphD(RHA1). The side-chain directions of Arg185 and Trp262 residues in the substrate binding pockets of these enzymes were different among these proteins, suggesting that these residues may take a conformational change during the catalytic cycles.
2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.
2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 A resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.
The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.
Title: Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63 Kasuga K, Habe H, Chung JS, Yoshida T, Nojiri H, Yamane H, Omori T Ref: Biochemical & Biophysical Research Communications, 283:195, 2001 : PubMed
A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].
Title: Cloning and characterization of genes involved in the degradation of dibenzofuran by Terrabacter sp. strain DBF63. Kasuga K, Nojiri H, Yamane H, Kodama T, Omori T Ref: J Ferment Bioeng, 84:387, 1997 : PubMed
Title: Cloning of genes involved in carbazole degradation of Pseudomonas sp. strain CA10: nucleotide sequences of genes and characterization of meta-cleavage enzymes and hydrolase Sato SI, Ouchiyama N, Kimura T, Nojiri H, Yamane H, Omori T Ref: Journal of Bacteriology, 179:4841, 1997 : PubMed
The DNA fragment encoding meta-cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp. strain CA10. DNA sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively. The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site. On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives. The putative polypeptide encoded by ORF3 was homologous with meta-cleavage compound hydrolases in other bacteria, and ORF3 was designated carC. The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic acid, the meta-cleavage compound of 3-methylcatechol. Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation pathway. These results suggest the possibility that CarC is a novel type of hydrolase.
Title: Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10 Sato SI, Nam JW, Kasuga K, Nojiri H, Yamane H, Omori T Ref: Journal of Bacteriology, 179:4850, 1997 : PubMed
Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.
Title: Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01 Habe H, Kasuga K, Nojiri H, Yamane H, Omori T Ref: Applied Environmental Microbiology, 62:4471, 1996 : PubMed
We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labb, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.
