Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

萘降解细菌的分离及其降解基因的分子检测

从污水处理厂的活性污泥和石油工业废水中各分离到24个降解萘的细菌分离株,提取这些分离株的总DNA,然后与各种萘降解基因杂交。结果表明,这2个来源的分离株在萘降解基因的种类上有明显不同。来自工业废水的分离株含有萘双加氧酶的铁硫蛋白大亚基基因nahAc,水杨醛脱氢酶基因nahF及其重复基因nahV,水杨酸羟化酶基因nahG及其重复基因nahU,儿茶酚2,3-双加氧酶基因nahH和儿茶酚1,2-双加氧酶基因catA,以及萘趋化蛋白基因nahY。来自活性污泥的分离株只含有nahAc、nahF、nahG和catA,不含有nahY、nahV、nahU和nahH。     

Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .     

Salicylate degradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains not involving the “Classical” <Emphasis Type="Italic">nah2</Emphasis> operon

Genetic systems for salicylate catabolism were analyzed in 12 strains of Pseudomonas putida, isolated from polluted soil samples collected in the Murmansk and Tula oblasts. All of the studied P. putida strains utilize salicylate in the ortho-pathway of catechol cleavage without employing the enzymes of the “classical” nah2 operon. The data demonstrates that salicylate degradation in the studied strains is performed with the involvement of the salicylate hydroxylase gene analogous to the nahU gene of strain P. putida ND6. New variants of salicylate hydroxylase genes nahG1 and nahU were found.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

The role of salicylic acid in the glutathione-mediated protection against photooxidative stress in rice

Salicylic acid (SA) is known to be an essential component responsible for disease resistance in dicotyledonous plants. In rice, however, tissue contains extremely high endogenous levels of SA that do not increase after pathogen infection, suggesting that the SA has other major functions in healthy leaves. Although involvement of SA in oxidative-stress response is known in some dicotyledonous plants, antioxidative role of SA in rice is obscure. In this study, we examined the involvement of SA in the protection against oxidative stress in rice, using transgenic plants expressing the bacterial nahG gene that encodes salicylate hydroxylase, an SA-degrading enzyme. In SA-deficient NahG rice, the glutathione pool size was constitutively diminished as compared with control plants. NahG seedlings showed a delayed development phenotype, an increased susceptibility to oxidative stress and they developed light-induced lesions in their leaves without pathogen infection. Conversely, treatment with an activator of the SA-mediated defense-signaling pathway, probenazole, increased the glutathione pool size and suppressed lesion formation. These results suggest that in rice, SA has an important role in the response to high-light-induced oxidative stress, through its regulatory effects on glutathione homeostasis.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.     

Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate-deficiency in tomato plants

Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens. Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase. Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants. In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death. The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves. These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv. tomato. However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants. This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development. These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion. We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.     

Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation.

In Arabidopsis, systemic acquired resistance against pathogens has been associated with the accumulation of salicylic acid (SA) and the expression of the pathogenesis-related proteins PR-1, PR-2, and PR-5. We report here the isolation of two nonallelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction-deficient (sid) mutants do not accumulate SA after pathogen inoculation and are more susceptible to both virulent and avirulent forms of Pseudomonas syringae and Peronospora parasitica. However, sid mutants are not as susceptible to these pathogens as are transgenic plants expressing the nahG gene encoding an SA hydroxylase that degrades SA to catechol. In contrast to NahG plants, only the expression of PR-1 is strongly reduced in sid mutants, whereas PR-2 and PR-5 are still expressed after pathogen attack. Furthermore, the accumulation of the phytoalexin camalexin is normal. These results indicate that SA-independent compensation pathways that do not operate in NahG plants are active in sid mutants. One of the mutants is allelic to eds5 (for enhanced disease susceptibility), whereas the other mutant has not been described previously.     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

Salicylate and catechol levels are maintained in nahG transgenic poplar

Metabolic profiling was used to investigate the molecular phenotypes of a transgenic Populus tremula x P. alba hybrid expressing the nahG transgene, a bacterial gene encoding salicylate hydroxylase that converts salicylic acid to catechol. Despite the efficacy of this transgenic approach to reduce salicylic acid levels in other model systems and thereby elucidate roles for salicylic acid in plant signaling, transgenic poplars had similar foliar levels of salicylic acid and catechol to that of non-transformed controls and exhibited no morphological phenotypes. To gain a deeper understanding of the basis for these observations, we analyzed metabolic profiles of leaves as influenced by transgene expression. Expression of nahG decreased quinic acid conjugates and increased catechol glucoside, while exerting little effect on levels of salicylic acid and catechol, the substrate and product, respectively, of the nahG enzyme. This suggests a biological role of elevated constitutive salicylic acid levels in Populus, in contrast to other plant systems in which nahG dramatically reduces salicylic acid levels.     

Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid

In plants carrying the NahG transgene, salicylate hydroxylase converts salicylic acid (SA) to catechol. Arabidopsis NahG plants are defective in non-host resistance to Pseudomonas syringae pv. phaseolicola strain 3121 (Psp), suggesting that resistance requires SA signaling. However, several mutants with defects in SA signaling, including eds1, pad4, eds5, sid2, and npr1, remain resistant to Psp, demonstrating that susceptibility of NahG plants is not due to absence of SA. SA synthesis is blocked in sid2NahG double mutants, but resistance to Psp is retained. Therefore, it must be the degradative action of NAHG on SA that causes the loss of resistance of NahG to Psp. Treatment of plants with catechol compromised Psp resistance suggesting that the effect of NahG on resistance results from catechol production. Application of catalase to NahG or catechol-treated wild-type plants partially restored resistance to Psp, suggesting that the deleterious effect of catechol results from inappropriate production of hydrogen peroxide. These results indicate that conclusions about SA requirements based solely on phenotypes of NahG plants should be re-evaluated.     

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.     

Novel aerobic 2-aminobenzoate metabolism. Nucleotide sequence of the plasmid carrying the gene for the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase in a denitrifying Pseudomonas sp.

Pseudomonas KB 740 degrades 2-aminobenzoate aerobically via a chimeric pathway which combines characteristics of anaerobic and aerobic aromatic metabolism. Atypically, 2-aminobenzoyl-CoA is an intermediate, and the activated aromatic acid is not only hydroxylated but also reduced to an alicyclic compound in a single step. The bacterial strain possesses a small plasmid, pKB 740, which carries all essential information of this new pathway. Its total nucleotide sequence was determined. It consists of 8280 bp and contains the genes for the two initial enzymes of the pathway; 2-aminobenzoate-CoA ligase catalyzes the activation of the aromatic acid, and the flavoenzyme 2-aminobenzoyl-CoA monooxygenase/reductase catalyzes the hydroxylation (monooxygenase activity) and subsequent reduction (reductase activity) of the aromatic ring of 2-aminobenzoyl-CoA. Furthermore, five open reading frames (ORF) possibly coding for polypeptides are on the plasmid. Putative promoter sequences were found for two of the ORF. A nucleotide sequence able to form a possible termination loop was located downstream of the gene for 2-aminobenzoyl-CoA monooxygenase/reductase. This gene consists of 2190 bases. The deduced amino acid sequence of the protein (730 residues; calculated molecular mass of the native 729-residue protein, 83,559 Da) contains a consensus sequence for an FAD-binding site at the N-terminus and a possible NAD(P)H-binding site approximately 150 amino acid residues apart from the N-terminus. The monooxygenase/reductase shows low sequence similarity to the flavoprotein salicylate hydroxylase. Functional and evolutionary aspects of this work are discussed.