Cloning and Characterization of the pnb Genes, Encoding Enzymes for 4-Nitrobenzoate Catabolism inPseudomonas putida TW3

ABSTRACT Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated withpnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3.

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Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82

Journal of Bioscience and Bioengineering ◽

10.1016/s1389-1723(99)80144-3 ◽

1999 ◽

Vol 87 (6) ◽

pp. 721-731 ◽

Cited By ~ 21

Author(s):

Noboru Takizawa ◽

Toshiya Iida ◽

Takashi Sawada ◽

Kazuhiro Yamauchi ◽

Yue-Wu Wang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Pseudomonas Putida ◽

Nucleotide Sequences ◽

Genes Encoding

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A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum

Microbiology ◽

10.1099/mic.0.29298-0 ◽

2006 ◽

Vol 152 (12) ◽

pp. 3517-3528 ◽

Cited By ~ 35

Author(s):

Miguel Balado ◽

Carlos R. Osorio ◽

Manuel L. Lemos

Keyword(s):

Gene Cluster ◽

Vibrio Anguillarum ◽

No Production ◽

Genetic Determinants ◽

Dihydroxybenzoic Acid ◽

Peptide Synthetase ◽

Genes Encoding ◽

In Trans ◽

Culture Supernatants

Vibrio anguillarum serotype O2 strains produce a catechol siderophore named vanchrobactin, which has been identified as N-[N′-(2,3-dihydroxybenzoyl)-arginyl]-serine. This work describes a chromosomal region that harbours the genetic determinants necessary for the biosynthesis of vanchrobactin. The authors have identified the genes involved in 2,3-dihydroxybenzoic acid (DHBA) biosynthesis (vabA, vabB and vabC) and activation (vabE), and a gene (vabF) encoding a non-ribosomal peptide synthetase, which is putatively involved in the assembly of the siderophore components. Also described are the identification and characterization of genes encoding a putative vanchrobactin exporter (vabS) and a siderophore esterase (vabH). In-frame deletion mutants in vabA, vabB, vabC, vabE, vabF and vabH were impaired for growth under conditions of iron limitation, and the analysis of culture supernatants by chrome azurol-S and cross-feeding assays showed almost no production of siderophores in any of the vabABCEF mutants. In addition, deletion mutations of vabA, vabB and vabC abolished production of DHBA, as assessed by chemical and biological analyses. Complementation of each mutant with the corresponding gene provided in trans confirmed the involvement of this gene cluster in the biosynthesis of DHBA and vanchrobactin in V. anguillarum strain RV22. Based on chemical and genetic data, and on published models for other catechol siderophores, a model for vanchrobactin biosynthesis is proposed.

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Characterization of Hybrid Toluate and Benzoate Dioxygenases

Journal of Bacteriology ◽

10.1128/jb.185.18.5333-5341.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5333-5341 ◽

Cited By ~ 5

Author(s):

Yong Ge ◽

Lindsay D. Eltis

Keyword(s):

Substrate Specificity ◽

Pseudomonas Putida ◽

Acinetobacter Calcoaceticus ◽

The Other ◽

Β Subunit ◽

Structural Determinants ◽

Α Subunit ◽

Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

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Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium Agrobacterium radiobacter Strain S2

Journal of Bacteriology ◽

10.1128/jb.182.21.6123-6129.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6123-6129 ◽

Cited By ~ 21

Author(s):

Matthias Contzen ◽

Andreas Stolz

Keyword(s):

Amino Acid Sequences ◽

Open Reading Frames ◽

Agrobacterium Radiobacter ◽

Sequence Alignments ◽

Degrading Bacterium ◽

Regulator Protein ◽

Putative Operon ◽

Genes Encoding ◽

Reading Frames

ABSTRACT The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacteriumAgrobacterium radiobacter strain S2 (DSMZ 5681). ThepcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strainAgrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

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Characterization of Oligosaccharides of the Lactosamine Series Derived from Keratan Sulfates by Tandem Mass Spectrometry

European Journal of Mass Spectrometry ◽

10.1255/ejms.338 ◽

2000 ◽

Vol 6 (2) ◽

pp. 193-203 ◽

Cited By ~ 10

Author(s):

Masayuki Kubota ◽

Keiichi Yoshida ◽

Akira Tawada ◽

Mamoru Ohashi

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Bond Cleavage ◽

The Other ◽

Negative Ion ◽

Ring Cleavage ◽

Tandem Mass ◽

Fragment Ions ◽

Glycosidic Bond Cleavage

Positive- and negative-ion fast-atom bombardment tandem mass spectrometry with collision-induced dissociation (FAB-CID-MS/MS) has been used in the characterization of di-and tetra-saccharides of the lactosamine series from keratan sulfates. FAB-CID-MS/MS of Galβ1-4GlcNAc (L1) exhibited strong fragment ions originating from ring cleavage at the reducing-terminal sugar moiety together with glycosidic bond-cleavage ions, whereas GlcNAcβ1-3Gal (K1) showed strong glycosidic bond-cleavage ions but no ring-cleavage ions. A series of ring-cleavage fragment ions was observed with members of the L-series which have free hydroxyl groups at the C1 and C3 positions. CID-MS/MS spectra of the [M + Na – SO3]+ ion ( m/z 406) from L2 and the [M + Na − 2SO3]+ ion ( m/z 406) from L4 were almost identical with the CID-MS/MS spectrum of the [M + Na]+ ion ( m/z 406) from L1, which indicated that the sugar skeletons of L2 and L4 are the same as that of L1. On the other hand, the CID-MS/MS spectrum of the [M + Na – SO3]+ ion ( m/z 508) from L4 did not resemble that of the [M + Na]+ ion ( m/z 508) from L2. The former showed peaks that were additional to the peaks in the latter. Since these extra peaks were accounted for on the basis of the structure of L3 [Galβ1(6S)-4GlcNAc, S = sulfate], the in-source loss of sulfate groups by ester exchange upon FAB ionization takes place in a dual manner; one reaction at the non-reducing terminal sugar to give L2 and the other at the reducing-terminal sugar to give L3. The CID-MS/MS spectra were characteristic for the tetrasaccharides L1-L1, L2-L2 and L4-L4 while in-source fragmentation confirms the component disaccharides of each tetrasaccharide. The structure of a tetrasaccharide trisulfate was confirmed as L2–L4 and not L4–L2 by CID-MS/MS. Negative-ion FAB-CID-MS/MS spectra of the sulfated di-and tetra-saccharides showed a pattern similar to that of the positive-ion spectra. Subtraction of the CID-MS/MS spectrum of the [M – H]− ion of L2 [Galβ1-4GlcNAc(6S)] from that of the [M – H – SO3]− ion of L4 [Gal(6S)β1-4GlcNAc(6S)] gave several specific ions whose origins were nicely explained on the basis of the structure of L3. The structure of a pentasaccharide consisting of N-acetylneuraminic acid and a tetrasaccharide trisulfate was confirmed, on the basis of FAB-CID-MS/MS, as NeuNAcα2-6L2-L4.

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Identification and characterization of 19 predicted Myb-family DNA binding proteins in Magnaporthe oryzae concerning growth, conidiation, and pathogenicity

10.1101/2021.12.28.474317 ◽

2021 ◽

Author(s):

Ya Li ◽

Xiuxia Zheng ◽

Mengtian Pei ◽

Mengting Chen ◽

Shengnan Zhang ◽

...

Keyword(s):

Stress Response ◽

Dna Binding ◽

Magnaporthe Oryzae ◽

Expression Profiles ◽

Dna Binding Proteins ◽

The Other ◽

Genes Encoding ◽

Identification And Characterization ◽

Different Levels

Genes encoding for proteins containing the DNA binding Myb domain have been suggested to be important in regulating development and stress response in eukaryotes, including fungi. Magnaporthe oryzae (teleomorph Pyricularia oryzae) is considered the most destructive pathogen of rice. We screen the M. oryzae genome for all genes encoding proteins containing Myb domains since these genes could be essential during pathogenesis. We found 19 genes Myb1-19. Only a few have previously been investigated, and only one has proven to be involved in pathogenesis. We tried to delete the other 18 genes and succeeded with all except 6, five of which could be essential. RT-qPCR showed that all 19 genes are expressed during pathogenesis, although at different levels and with different expression profiles. To our surprise, only deletions of the genes encoding proteins MoMyb2, MoMyb13, and MoMyb15 showed growth, conidiation, and infection phenotypes, indicating that they are essential on their own during infection. This lack of phenotypes for the other mutants surprised us, and we extended the analysis to look for expression co-regulation and found 5 co-regulated groups of predicted proteins with Myb-domains. We point to likely compensatory regulations of the other Myb-family genes hiding the effect of many deletions. Further studies of the Myb-family genes are thus of interest since revealing the functions of these genes with a possible effect on pathogenicity since these could be targets for future measures to control M. oryzae in rice.

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