Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3

A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).     

Resolution of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 into three components

Extracts of Pseudomonas sp. strain CBS3 grown with 4-chlorobenzoate as sole carbon source contained an enzyme that converted 4-chlorobenzoate to 4-hydroxybenzoate. This enzyme was shown to consist of three components, all necessary for the reaction. Component I, which had a molecular weight of about 3,000, was highly unstable. Components II and III were stable proteins with molecular weights of about 86,000 and 92,000.     

Resolution of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 into three components.

Extracts of Pseudomonas sp. strain CBS3 grown with 4-chlorobenzoate as sole carbon source contained an enzyme that converted 4-chlorobenzoate to 4-hydroxybenzoate. This enzyme was shown to consist of three components, all necessary for the reaction. Component I, which had a molecular weight of about 3,000, was highly unstable. Components II and III were stable proteins with molecular weights of about 86,000 and 92,000.     

Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.

Two genes, xynA and xynB, encoding xylanases from Paenibacillus sp. KCTC 8848P were cloned and expressed in Escherichia coli, and their nucleotide sequences were determined. The xylanases of E. coli transformants were released into the extracellular culture fluid in the absence of xylan. The structural gene of xynA 636 bp, encoded a protein of 212 amino acids, while the xynB gene consisted of 951 bp open reading frame for a protein of 317 amino acids. The amino acid sequence of the xynAgene showed 83% similarity to the xylanase of Aeromonas caviae, and belonged to the family 11 glycosyl hydrolases. The deduced amino acid sequence of the xynB gene, however, showed 51% similarity to the xylanase of Rhodothermus marinus, and belonged to the family 10 glycosyl hydrolases.     

Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.     

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.     

Nucleotide sequences of the genes for two distinct cephalosporin acylases from a Pseudomonas strain.

Two genes, acyI and acyII, for distinct cephalosporin acylases from Pseudomonas sp. strain SE83 (A. Matsuda, K. Matsuyama, K. Yamamoto, S. Ichikawa, and K.I. Komatsu, J. Bacteriol. 169:5815-5820, 1987) were sequenced. Each sequence contained a single open reading frame for two nonidentical subunits, predicting a common precursor. Some homologies at the amino acid level were found between the acyII-encoded enzyme, but not the acyI-encoded one, and other related acylases.     

Oxidation and dehalogenation of 4-chlorophenylacetate by a two-component enzyme system from Pseudomonas sp. strain CBS3

In cell-free extracts from Pseudomonas sp. strain CBS3 the conversion of 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate was demonstrated. By Sephacryl S-200 chromatography two protein fractions, A and B, were obtained which both were essential for enzyme activity. Fe2+ and NADH were cofactors of the reaction. NADPH also activated the enzyme, but less effectively than NADH. FAD had no influence on enzyme activity. 4-Hydroxyphenylacetate, 4-chloro-3-hydroxyphenylacetate, and 3-chloro-4-hydroxyphenylacetate were poor substrates for the enzyme, suggesting that these substances are not intermediates of the reaction. We therefore suggest that the reaction proceeds via a dioxygenated intermediate.     

Initial characterisation of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

Extracts of Pseudomonas sp. CBS3 converted 4-chlorobenzoate into 4-hydroxybenzoate. The enzyme responsible for this conversion was enriched by ammonium sulphate fractionation (30–60% saturation, 1.3-fold). The optimum conditions for the reaction were 30–35°C and pH 7–7.5. The enzyme was activated by Mn²⁺ (1 mM final concentration) up to 120-fold, and by Co²⁺ (1 mM final concentration) up to 60-fold. Other divalent ions had no effect. EDTA inhibited the enzyme. 4-Bromobenzoate and 4-iodobenzoate were substrates for the enzyme, but 4-fluorobenzoate was not converted.     

The nucleotide sequences of two leghemoglobin genes from soybean.

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes correspond to leghemoglobin C2 and leghemoglobin C3, respectively.     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Purification and characterization of 2-halocarboxylic acid dehalogenase II from Pseudomonas spec. CBS 3.

2-Halocarboxylic acid dehalogenase II from Pseudomonas spec. CBS 3 (EC 3.8.1.2), which had been cloned in E. coli Hb 101 was purified to electrophoretic homogeneity from crude extracts of E. coli Hb 101 clone 1164. Ammonium sulfate fractionation and three subsequent chromatographic purification steps yielded a pure enzyme in a 230-fold enrichment. The relative molecular masses as determined by gelfiltration on Superose 12 and SDS-polyacrylamide gel electrophoresis were 64,000 Da for the holoenzyme and 29,000 Da for the subunit. The isoelectric point, determined by isoelectric focusing, was at pH 6.2. Substrate specificity towards chlorinated and brominated substrates was limited to short chain monosubstituted 2-halocarboxylic acids. Fluorocompounds were not converted. The reaction proceeded best at a pH above 9.5 and at a reaction temperature of 40-45 degrees C.     

Complete genome sequences of Methylophaga sp. strain JAM1 and Methylophaga sp. strain JAM7

Methylophaga sp. strains JAM1 and JAM7 have been isolated from a denitrification system. Strain JAM1 was the first Methylophaga strain reported to be able to grow under denitrifying conditions. Here, we report the complete genome sequences of the two strains, which allowed prediction of gene clusters involved in denitrification in strain JAM1.