High-density sampling of a bacterial operon using mRNA differential display.

We have implemented a simplified high throughput approach to differential display in order to identify transcriptionally regulated genes in bacteria. In contrast with the few previous applications of differential display to prokaryotes, we use a large number of primers which allows for a high-density sampling of the mRNA population and the identification of many differentially amplified DNA fragments. From the overlap of these short sequences, long contiguous sequences that encode several genes can be assembled. The multiplicity of sampling provides a strong indication that the genes identified are indeed differentially regulated. As a test case, we looked for the genes involved in the degradation of 2,4-dinitrophenol (2,4-DNP) in a Rhodococcus erythropolis strain, HL PM-1. In this experiment a long polycistronic mRNA was sampled repeatedly. The induction of these genes by 2,4-DNP was confirmed by dot blot analysis and two of them were confirmed to be involved in the degradation of 2,4-DNP. This work shows that mRNA differential display is an important tool for the identification of metabolic genes in prokaryotes.

Genetic and biochemical characterization of an enantioselective amidase from Agrobacterium tumefaciens strain d3.

An enantioselective amidase was purified to homogeneity from Agrobacterium tumefaciens d3. The enzyme has a molecular mass of about 490000 Da and is composed of identical subunits with a molecular mass of about 63000 Da. The purified enzyme converted racemic 2-phenylpropionamide to the corresponding S-acid with an enantiomeric excess (ee) value >95% at almost 50% conversion of the racemic amide. The purified enzyme was digested with trypsin and the amino acid sequences of the N terminus and different tryptic peptides determined. These amino acid sequences were used to clone the encoding gene. Finally, a 9330 bp DNA fragment was sequenced and the amidase gene identified. The deduced amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. No indications of a structural coupling of the amidase gene with the genes for a nitrile hydratase could be found on the cloned DNA fragment. The amidase gene was encoded by an approximately 500 kb circular plasmid in A. tumefaciens d3. The amidase was heterologously expressed in Escherichia coli and, as well as 2-phenylpropionamide, was shown to hydrolyse alpha-chloro- and alpha-methoxyphenylacetamide and 2-methyl-3-phenylpropionamide highly enantioselectively. Some amino acids within a highly conserved region common amongst all known enantioselective amidases ('amidase signature') were changed by site-specific mutagenesis and significant changes in the relative activities with different amides observed.

Converging catabolism of 2,4,6-trinitrophenol (picric acid) and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A.

Initial F420-dependent hydrogenation of 2,4,6-trinitrophenol (picric acid) generated the hydride sigma-complex of picrate and finally the dihydride complex. With 2,4-dinitrophenol the hydride sigma-complex of 2,4-dinitrophenol is generated. The hydride transferring enzyme system showed activity against several substituted 2,4-dinitrophenols but not with mononitrophenols. A Km-value of 0.06 mM of the hydride transfer for picrate as substrate was found. The pH optima of the NADPH-dependent F420 reductase and for the hydride transferase were 5.5 and 7.5, respectively. An enzymatic activity has been identified catalyzing the release of stoichometric amounts of 1 mol nitrite from 1 mol of the dihydride sigma-complex of picrate. This complex was synthesized by chemical reduction of picrate and characterized by 1H and 13C NMR spectroscopy. The hydride sigma-complex of 2,4-dinitrophenol has been identified as the denitration product. The nitrite-eliminating activity was enriched and clearly separated from the hydride transferring enzyme system by FPLC. 2,4-Dinitrophenol has been disproven as a metabolite of picrate (Ebert et al. 1999) and a convergent catabolic pathway for picrate and 2,4-dinitrophenol with the hydride sigma-complex of 2,4-dinitrophenol as the common intermediate has been demonstrated.

Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1.

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the Km and Vmax values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate. The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate 1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.

Catalytic properties of the 3-chlorocatechol-oxidizing 2, 3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas sp. strain BN6.

The 2,3-dihydroxybiphenyl dioxygenase from Sphingomonas sp. strain BN6 (BphC1-BN6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of different substrates and the effects of various inhibitors on BphC1-BN6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid. Cell extracts containing C23O-mt2 or BphC2-BN6 converted the relevant substrates with an almost constant rate for at least 10 min, whereas BphC1-BN6 was inactivated significantly within the first minutes during the turnover of all substrates tested. Furthermore, BphC1-BN6 was much more sensitive than the other two enzymes to inactivation by the Fe(II) ion-chelating compound o-phenanthroline. The reason for inactivation of BphC1-BN6 appeared to be the loss of the weakly bound ferrous ion, which is the cofactor in the catalytic center. A mutant enzyme of BphC1-BN6 constructed by site-directed mutagenesis showed a higher stability to inactivation by o-phenanthroline and an increased catalytic efficiency for the conversion of 2,3-dihydroxybiphenyl and 3-methylcatechol but was still inactivated during substrate oxidation.

Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134.

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

Function of coenzyme F420 in aerobic catabolism of 2,4, 6-trinitrophenol and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A.

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these pi-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.

Hydride-Meisenheimer complex formation and protonation as key reactions of 2,4,6-trinitrophenol biodegradation by Rhodococcus erythropolis.

Biodegradation of 2,4,6-trinitrophenol (picric acid) by Rhodococcus erythropolis HLPM-1 proceeds via initial hydrogenation of the aromatic ring system. Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic sigma-complex) of picric acid and its protonated form under physiological conditions. These complexes are key intermediates of denitration and productive microbial degradation of picric acid. For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time. Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid. This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite. Cell extracts of R. erythropolis HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol. Picric acid is used as the sole carbon, nitrogen, and energy source by R. erythropolis HLPM-1.

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a Bamberger rearrangement.

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.

Isolation of a bacterial strain with the ability to utilize the sulfonated azo compound 4-carboxy-4'-sulfoazobenzene as the sole source of carbon and energy.

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4'-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of "Hydrogenophaga palleronii" S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4'-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.

A new 4-nitrotoluene degradation pathway in a Mycobacterium strain.

Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (lambda max, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage.

Initial reductive reactions in aerobic microbial metabolism of 2,4,6-trinitrotoluene.

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.

Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6.

The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture. Physiological interactions between Hydrogenophaga palleronii S1 and Agrobacterium radiobacter S2.

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B12. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.

Purification and properties of an amidase from Rhodococcus erythropolis MP50 which enantioselectively hydrolyzes 2-arylpropionamides.

An enantioselective amidase from Rhodococcus erythropolis MP50 was purified to homogeneity. The enzyme has a molecular weight of about 480,000 and is composed of identical subunits with molecular weights of about 61,000. The NH2-terminal amino acid sequence was significantly different from previously published sequences of bacterial amidases. The purified amidase hydrolyzed a wide range of aliphatic and aromatic amides, The highest enzyme activities were found with amides carrying hydrophobic residues, such as pentyl or naphthoyl. The purified enzyme converted racemic 2-phenylpropionamide, naproxen amide [2-(6-methoxy-2-naphthyl) propionamide], and ketoprofen amide [2-(3'-benzoylphenyl)propionamide] to the corresponding S-acids with an enantiomeric excess of >99% and an almost 50% conversion of the racemic amides. The enzyme also hydrolyzed different alpha-amino amides but without significant enantioselectivity.

A dynamic river model for biodegradability studies: investigations with selected aromatic compounds at low concentrations and comparison with aquatic batch tests.

The objective of this publication is to present a new dynamic aerobic biodegradation test method simulating a river. A laboratory cascade test system and standardized batch shake flask tests were used for biodegradation studies with the non-volatile and non-sorbing model compounds 2,4-dinitrophenol, naphthalene-1-sulphonic acid and sulphanilic acid. To be closer to the often very low concentrations of substances in the environment the concentrations of the compounds used were standard test concentrations and lower. 14C labelled compounds were measured at 50 micrograms/l, capillary electrophoresis at 5000 micrograms/l and the removal of dissolved organic carbon at 50000 micrograms/l. The test results obtained confirmed the known ultimate biodegradability of the test compounds and showed that biodegradation degrees, rates and degradation durations depended on the test systems, the concentrations of test compounds and the inocula. The river model is a suitable simulation test for natural dynamic surface waters which can be used to perform biodegradability studies at low test concentrations if adequate analytical tools, preferably radioactive-labelled substances, are available.

Degradation of benzene 1,3-disulfonate by a mixed bacterial culture.

A benzene 1,3-disulfonate degrading mixed bacterial culture was isolated from the River Elbe downstream of Hamburg. The mixed culture was composed of five different bacterial strains. None of these strains grew in axenic culture with benzene 1,3-disulfonate as sole source of carbon and energy. In the presence of 4-nitrocatechol, resting cells of the mixed culture converted benzene 1,3-disulfonate to catechol 4-sulfonate. Experiments with cell-free extracts demonstrated that catechol 4-sulfonate was further metabolized via 3-sulfomuconate and 4-carboxymethyl-4-sulfobut-2-en-4-olide.

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6.

An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3- and 4-chlorocatechol, and 3- and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.

Aerobic biodegradation of 3-aminobenzoate by gram-negative bacteria involves intermediate formation of 5-aminosalicylate as ring-cleavage substrate.

The aerobic metabolism of 3-aminobenzoate by bacteria was studied. Bacterial strains degrading 3-aminobenzoate were obtained by enrichment with 3-aminobenzoate (strain Ia3) or 5-aminosalicylate (strains BN9 and 5AS1). During growth with 3-aminobenzoate, strain Ia3 and strain 5AS1 transiently accumulated 5-aminosalicylate in the culture broth. In the presence of inhibitors of 5-aminosalicylate 1,2-dioxygenase, resting cells of all three strains converted 3-aminobenzoate to stoichiometric amounts of 5-aminosalicylate. 5-Aminosalicylate 1,2-dioxygenase activity was induced in all strains after growth with 3-aminobenzoate or 5-aminosalicylate, but not after growth in complex media.

Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate.

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro- and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Alcaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans-4-carboxymethylene-but-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbon-chlorine bond during their divergence from normal muconate cycloisomerases.