Walter Reineke

Engineering bacteria for bioremediation.

The treatment of environmental pollution by microorganisms is a promising technology. Various genetic approaches have been developed and used to optimize the enzymes, metabolic pathways and organisms relevant for biodegradation. New information on the metabolic routes and bottlenecks of degradation is still accumulating, enlarging the available toolbox. With molecular methods allowing the characterization of microbial community structure and activities, the performance of microorganisms under in situ conditions and in concert with the indigenous microflora will become predictable.

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.

Chemical structure and biodegradability of halogenated aromatic compounds substituent effects on 1,2-dioxygenation of benzoic acid

Dioxygenation of substituted benzoic acids by whole cells of 3-chlorobenzoate-utilizing Pseudomonas sp. B 13, benzoate-induced cells of Alcaligenes eutrophus B 9 and toluate-grown cells of Pseudomonas putida mt-2 was examined. Electron-attracting substituents like halogen decreased the reaction rates of benzoate 1,2-dioxygenation. Dioxygenation of substituted benzoic acids by P. putida mt-2 was mostly undisturbed by steric effects of the substituents. Good correlation resulted between the log Vrel values and the Hammett substituent constant sigma. In contrast the reaction rates of dioxygenation by Pseudomonas sp. B 13 and A. eutrophus were decreased predominantly by steric effects of substituents. A non-polar reaction mechanism of benzoate 1,2-dioxygenation is discussed. Results from inhibition studies demonstrate high stereospecificities for the 1,2-dioxygenation by Pseudomonas sp. B 13 of benzoic acids with substituents in ortho- or para-position. In the case of P. putida mt-2 steric handrance by substituents was observed only with orth-substituted benzoic acids. Stereospecificities of the benzoate 1,2-dioxygenation by Pseudomonas sp. B 13 and P. putida mt-2 are illustrated schematically.

Microbial metabolism of chloroanilines: enhanced evolution by natural genetic exchange

Abstract2-, 3-, and 4-chloroaniline degrading bacteria were obtained by natural genetic exchange between an aniline or toluidine degrading Pseudomonas strain and the chlorocatechol assimilating Pseudomonas sp. B13. Hybrid organisms were isolated through cocultivation of the parent strains in the chemostat as well as through conjugation on solid media in presence of chloroanilines as the selective substrates. Biochemical analysis of the gene products in the hybrid strains clearly showed that the genes coding for the aniline dioxygenase or the genes for the chlorocatechol assimilatory sequence had been transferred.

Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates, metabolites in the degradation of chloroaromatic compounds.

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.

Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.

Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 +/- 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 +/- 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.

Comparative Specificities of Two Evolutionarily Divergent Hydrolases Involved in Microbial Degradation of Polychlorinated Biphenyls

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701-15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD(P6) hydrolyzed HOPDA with a k(cat)/K(m) of 1.62 (+/- 0.03) x 10(7) M(-1) s(-1) (100 mM phosphate [pH 7.5], 25 degrees C), which is within 70% of that of BphD(LB400). BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD(LB400). These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 μM) and 3,4-CBA (50 μM). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.