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Applied and Environmental Microbiology, April 2005, p. 2199-2202, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2199-2202.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Opposite Enantioselectivities of Two Phenotypically and Genotypically Similar Strains of Pseudomonas frederiksbergensis in Bacterial Whole-Cell Sulfoxidation

Waldemar Adam,^1,2 Frank Heckel,³ Chantu R. Saha-Möller,¹ Marcus Taupp,³ Jean-Marie Meyer,⁴ and Peter Schreier^3*

Institute of Organic Chemistry,¹ Institute of Food Chemistry, University of Würzburg, Würzburg, Germany,³ Department of Chemistry, Facundo University of Puerto Rico, Rio Piedras, Puerto Rico,² Laboratoire de Microbiologie et Génétique, Université Louis Pasteur, Strasbourg, France⁴

Received 19 August 2004/ Accepted 12 November 2004

Top Abstract Introduction Nucleotide sequence accession... References

 
Soil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strains that preferentially produced the S-configured sulfoxide enantiomer were isolated. Surprisingly, one bacterial strain, genotypically and phenotypically characterized as Pseudomonas frederiksbergensis, selectively gave the R enantiomer. The finding that two apparently identical organisms displayed opposite enantioselectivities is novel for non-genetically modified organisms.

Top Abstract Introduction Nucleotide sequence accession... References

 
Sulfoxides are versatile and convenient chiral building blocks in asymmetric synthesis. In particular, enantiomerically pure sulfoxides play an important role either as chiral building blocks or as stereo-directing groups (20). The value of sulfoxide functionality is further illustrated by their diverse biological activities and pharmaceutical uses. In most cases, only one enantiomer is responsible for the desired biological activity.

Enzyme-catalyzed asymmetric sulfoxidations have been developed in recent years for the preparation of chiral sulfoxides with an enantiomeric excess (ee value; for a definition, see Table 1) up to 100% (2, 3, 5, 9, 10, 17). Especially peroxidases efficiently catalyze the enantioselective sulfoxidation of a variety of sulfides (4, 11, 23). Several whole-cell systems have also been successfully employed in biocatalytic sulfoxidation; among them, some bacterial strains hold great promise for the development of environmentally benign, efficient asymmetric sulfoxidation (16, 19, 22). For example, we have shown that the topsoil bacterium Pseudomonas frederiksbergensis, recently isolated at a coal gasification site in Frederiksberg, Copenhagen, Denmark (6, 18), catalyzes the asymmetric oxidation of sulfides to enantiomerically enriched sulfoxides (1). Since the hitherto known isolated enzymes and bacterial whole-cell systems preferentially produce only one of the two enantiomers (either R or S), finding bacteria delivering both enantiomers is a promising goal. So far, only one example appears to be known, namely, a Pseudomonas putida strain, which produces one enantiomer of the sulfoxide in excellent enantioselectivity, whereas a genetically engineered mutant of this bacterium and the native strain afford the opposite enantiomers (8).

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TABLE 1. Enantioselectivity in the asymmetric oxidation of phenyl methyl sulfide (Ia) to the corresponding sulfoxide IIa by isolated soil microorganisms

To find soil microorganisms for asymmetric sulfoxidation, a sulfide-mediated screening was carried out. Strains were isolated from soil samples taken in Geiselwind, Germany, by treatment with 10 µl of phenyl methyl sulfide (Ia) per plate on minimal agar, with glucose as the carbon source. After isolation of the sulfur-adapted microorganisms, these pure cultures were used for standard oxidations of phenyl methyl sulfide (Ia) as substrate to the enantiomerically enriched phenyl methyl sulfoxide (IIa), as displayed in Fig. 1. Few strains possessed acceptable efficiency for asymmetric sulfoxidation (Table 1) and were therefore characterized by partial sequencing of the 16S rRNA gene. The preparation of intact bacterial cells for PCR was performed as described in the literature (24). Single-stranded DNA was sequenced with internal primers by using the Taq-cycle DyeDeoxy Terminator method. Finally, a homology search was performed (15) with the acquired sequence data against the European Molecular Biology Laboratory (EMBL) database (14). The results presented in Table 1 reveal that only one strain, namely, strain 33 (Table 1, entry 3), produced the R enantiomer of phenyl methyl sulfoxide (IIa). All other strains produced mainly the S enantiomer; indeed, in the case of Arthrobacter aurescens (Table 1, entry 5), an enantiomeric excess (ee) of 92% (S) was achieved. The identified Arthrobacter chlorophenolicus strain (Table 1, entry 1), as well as the P. putida, Pseudomonas fluorescens, and Pseudomonas aureofaciens strains (Table 1, entries 2, 4, and 6), converted the substrate phenyl methyl sulfide (Ia) to the corresponding sulfoxide IIa in ee values of 50 to 60%.

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FIG. 1. Enantioselective sulfoxidation of phenyl methyl sulfide (Ia) by isolated soil bacteria.

Strain 33 was characterized by standard phenotypical methods. Therefore, its appearance (by microscopic study, which included Gram stain) and behavior (optimal growth temperature and pH value) were assessed. The motility and the oxidase and catalase activities were positive. The classical API 20 NE test (bioMerieux, Nürtingen, Germany) was performed to compare strain 33 with the commercially available strain P. frederiksbergensis DSM 13022, which was purchased from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). This method combines 8 conventional tests and 12 assimilation tests for the identification of nonfastidious, gram-negative rods that belong to the Enterobacteriaceae, e.g., Pseudomonas. All observed color reactions and assimilation tests were identical for strain 33 and P. frederiksbergensis DSM 13022; for example, potassium nitrate was reduced to nitrite by both strains. The color reactions for detecting indole production with tryptophan as substrate and acidification with glucose were negative. The various tests for enzyme activity were negative, including arginine dihydrolase, urease, ß-glucosidase and protease, and ß-galactosidase. The assimilation tests with standard substrates as carbon sources (glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, gluconate, caprate, malate, citrate, and phenyl acetate) were positive; only maltose and adipate were negative. Finally, tetramethyl-p-phenylenediamine yielded a positive cytochrome oxidase reaction. To confirm the phenotypic identity between strain 33 and P. frederiksbergensis DSM 13022, the cellular fatty acid profile of strain 33 was determined by the identification service of the DSMZ and shown to be typical for Pseudomonas organisms. Altogether, the phenotypical characterization identified strain 33 as P. frederiksbergensis.

A direct comparison of the 16S rRNA partial sequences indicated that both bacteria belong to the species P. frederiksbergensis. The sequences of P. frederiksbergensis DSM 13022 and strain 33 are identical for 441 base pairs, which strongly suggests that the 16S rRNA genes of the two strains are phylogenetically the same.

For further verification, siderophore typing, a powerful tool for the identification of even nonfluorescent pseudomonads, was used. This newly developed technique (21) measures the iron uptake of the siderophores located in the P. frederiksbergensis strains by means of a cross-incorporation comparison. Siderophore typing experiments were carried out according to the previously described procedure (21) in duplicate with strain 33 by employing the commercially available P. frederiksbergensis DSM 13022 and P. frederiksbergensis JAJ 28 from J.-M. Meyer's collection (21). The siderophore system of P. frederiksbergensis DSM 13022 accomplished an iron uptake of 95% efficiency compared to that of strain 33. Conversely, strain 33 has a siderophore-mediated iron uptake of 99% efficiency with regard to P. frederiksbergensis DSM 13022. Identical results were obtained in comparison with P. frederiksbergensis JAJ 28. Thus, our strain 33 possesses the same siderotype as P. frederiksbergensis DSM 13022 and JAJ 28; accordingly, strain 33 belongs to the same siderovar, namely that of P. frederiksbergensis.

In order to determine substrate selectivity and the effects of variation in the sulfur substituent on the sense (R versus S) and extent (enantiomeric excess) of the selectivity of the sulfoxidation reactions, a series of sulfides (Ia to Ih) were tested in whole-cell cultures. The bacterial sulfoxidation experiments were carried out in 300-ml Erlenmeyer flasks, which were charged with 100 ml of Dworkin mineral nutriment solution (13) and 375 µl of Pseudomonas trace element solution (7). This medium was sterilized at 121°C for 16 min prior to the addition of 750 µl of 50% sterile glucose solution. After inoculation with bacterial material, the culture was pregrown for 24 h and subsequently shaken in the presence of 100 µmol of the appropriate sulfide (analytical grade; obtained from Fluka [Seelze, Germany], Sigma-Aldrich [Taufkirchen, Germany], or Lancaster [Morecambe, United Kingdom]) for 18 h at 30°C and 120 rpm. Subsequently, the bacterial cells were treated by ultrasound to release any sulfoxides retained in the cells. After centrifugation (9,000 rpm for 15 min in a Beckman J2-21 centrifuge equipped with a JA 10 rotor), the bacterial suspension was successively extracted twice with 100 ml of ethyl ether and 100 ml of dichloromethane (the solvents were freshly distilled before use). Finally, the products were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The chiral analysis of the products was performed by a multidimensional GC-MS combination on a 2,3-diethyl-6-t-butyldimethylsilyl-ß-cyclodextrin column. The absolute configurations of the sulfoxides were determined by comparison with the authentic samples, prepared by enzymatic sulfoxidation with chloroperoxidase according to the previously reported procedure (11), and with data from the literature (12).

The results for P. frederiksbergensis strain 33 and for the commercially available P. frederiksbergensis DSM 13022 are compared in Table 2. The yields ranged from 4 to 48% (data not shown) and were not optimized, since the emphasis of this work lies on the opposite enantioselectivities of the P. frederiksbergensis strains. As for the specific results, phenyl methyl sulfide (Ia) was sulfoxidized in an enantiomeric excess (ee) of 91% in favor of the S enantiomer by DSM 13022, whereas strain 33 produced the R enantiomer with an ee value of 62% (Table 2, entry 1). Introduction of a methyl group in the para position (Table 2, entry 2, Ib) afforded the enantiopure S-sulfoxide for DSM 13022, and again, mainly the R enantiomer was produced by strain 33. To assess the effect of the alkyl side chain on enantioselectivity, the n-propyl derivative Ic was employed as a substrate. A significantly lower enantioselectivity (81% ee in favor of S) for DSM 13022 and a poor ee value (that is, <5%) for strain 33 compared to the methyl derivative Ia were observed (Table 2, entry 3). Thus, the chain length has a pronounced effect on the enantioselectivity of sulfoxidation by both P. frederiksbergensis strains. Moreover, a branched alkyl chain dramatically lowers the substrate acceptance; for example, the isopropyl derivative Id was barely consumed by both bacterial strains (Table 2, entry 4). The enantioselectivity of strain 33, in contrast to that of DSM 13022, depends on the electronic properties of the para substituents. The oxidation of the electron-donating methoxy derivative Ie by strain 33 yielded the R sulfoxide with the highest ee value of 89% (Table 2, entry 5), while the electron-withdrawing chloro derivative If afforded the same sulfoxide enantiomer in 56% ee (Table 2, entry 6). In contrast, DSM 13022 oxidized both derivatives in ee values of 96 to 99% with a preference for the S enantiomer. Not only the alkyl aryl sulfides Ia to If but also dialkyl sulfides, e.g., cyclohexyl methyl sulfide (Ig), were enantioselectively sulfoxidized by DSM 13022 (70% S) and strain 33 (23% R), as shown in Table 2, entry 7. In contrast, the benzyl methyl sulfide (Ih) was not accepted by either strain (Table 2, entry 8).

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TABLE 2. Enantioselective oxidation of sulfides I to sulfoxides II by P. frederiksbergensis DSM 13022 and P. frederiksbergensis strain 33

The fact that the standard characterization method (partial sequencing of the 16S rRNA) exposed that strain 33 exclusively biosynthesizes the R-configured enantiomer in the sulfoxidation of aryl alkyl sulfides, in contrast to the commercially available strain DSM 13022, which preferentially expresses the S-configured sulfoxide, obliged extensive additional experimental probing into this novel phenomenon. This extensive examination included color reactions, assimilation tests, fatty acid profiles, and the powerful identification tool known as siderophore typing, which all confirmed a strong similarity between the two strains described. Herein, by all of the tested methods, P. frederiksbergensis strain 33 and P. frederiksbergensis DSM 13022 are similar in nature. Thus, for the first time two natural, genetically unmanipulated P. frederiksbergensis strains show inverse senses of enantioselectivity in the sulfoxidation of a variety of sulfide substrates. To date, only one other example of such a phenomenon in enantioselectivity has been reported (8), namely, the sulfoxidation by a wild-type Pseudomonas putida strain that contains naphthalene dioxygenase. In this case, the S-configured sulfoxide is preferably produced, whereas the recombinant Pseudomonas putida strain with toluene dioxygenase selects the R-configured enantiomer. It should also be emphasized that these different enantioselectivities between a native strain and a genetically modified Pseudomonas strain were not observed for all substrates used in the study (8). Of course, both P. frederiksbergensis strains investigated here must differ at the molecular level in order to express opposite sulfoxide enantiomers; thus, presumably the amino acid sequence for the enzyme responsible for the asymmetric sulfoxidation with opposite enantioselectivities must be distinct. Consequently, it should be a priority to determine the structure of the sulfoxidizing enzymes and their amino acid sequences. We speculate that the naphthalene dioxygenase and toluene dioxygenase enzymes described for the P. putida bacteria (8) might be involved in the sulfoxidation reactions of the P. frederiksbergensis strains described herein. Tentatively, the present results may be explained either in terms of a mutation within the enzyme responsible for enantioselective sulfoxidation or by the existence of two (or more) competing enzyme activities. We anticipate that our novel findings will initiate an intensive search for other such natural microorganisms.

Top Abstract Introduction Nucleotide sequence accession... References

 
The 16S rRNA partial sequence has been submitted to GenBank with the accession number AY817058. P. frederiksbergensis strain 33 was deposited at DSMZ under the DSMZ number 16916.

 
We are grateful to U. Vogel of the Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany, for the genotypical characterization (16S rRNA analysis) of the isolated soil bacteria.

The Deutsche Forschungsgemeinschaft (SFB 347) and the Fonds der Chemischen Industrie are thanked for generous financial support.

 
* Corresponding author. Mailing address: Institute of Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. Phone: 49-(0)-931-8885480. Fax: 49-(0)-931-8885484. E-mail: schreier{at}pzlc.uni-wuerzburg.de.

Top Abstract Introduction Nucleotide sequence accession... References

 

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Applied and Environmental Microbiology, April 2005, p. 2199-2202, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2199-2202.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Adam, W. Articles by Schreier, P. Search for Related Content PubMed PubMed Citation Articles by Adam, W. Articles by Schreier, P. Agricola Articles by Adam, W. Articles by Schreier, P.