List of journal articles Wouter Duetz

35) Brooks SJ, Doyle EM, Hewage C, Malthouse JPG, Duetz W, O'Connor KE (2004)

Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 64: 486-492 MAY 2004

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

34) Duetz, WA, Witholt, B. (2004)

Oxygen transfer by orbital shaking of square vessels and deepwell microtiter plates of various dimensions

BIOCHEMICAL ENGINEERING JOURNAL 17: 181–185

Oxygen transfer rates (OTRs) during orbital shaking were measured for differently sized polypropylene vessels that were either square

or round in the horizontal plane, using an enzymatic method involving glucose oxidase, horseradish peroxidase (HRP), and 2,2-azino-bis

3-ethybenz-thiazoline-6-sulfonic acid (ABTS). In comparison to non-shaking conditions (3.2 mmol O2 l1 h1), OTRs in 4mm × 4mm

vessels (corresponding to wells in 384-square deepwell microtiter plates) at a working volume of 0.125 ml could only be significantly

enhanced at 300 rpm if a shaking diameter of 50mm was applied (25 mmol O2 l1 h1) instead of 25mm (6.7 mmol O2 l1 h1). Larger

square vessels (18mm×18mm and 50mm×50 mm) yielded high oxygen transfer rates and a regular shaking pattern, demonstrating that

vessels in this size range could be a viable, space-efficient, alternative to baffled or unbaffled Erlenmeyer shaking flasks. Round vessels

(internal diameter 6.6 mm) resulted in OTRs that were approximately 50% of those measured for square vessels in the same size range.

33) van Beilen JB, Li Z, Duetz WA, Smits THM, Witholt B (2003)

Diversity of alkane hydroxylase systems in the environment

OIL & GAS SCIENCE AND TECHNOLOGY-REVUE DE L INSTITUT FRANCAIS DU PETROLE 58 (4): 427-440

The first step in the aerobic degradation of alkanes by bacteria, yeasts, and fungi is catalyzed by oxygenases. These enzymes, which introduce oxygen atoms derived from molecular oxygen into the alkane substrate, play an important role in oil bioremediation and in the cometabolic degradation of compounds such as trichloroethylene and fuel oxygenates. In addition, they are useful biocatalysts and important models for a difficult chemical reaction: the regio- and stereospecific activation of C-H bonds. Several unrelated enzyme classes catalyze the oxidation of alkanes. Alkane-degrading yeast strains contain multiple alkane hydroxylases belonging to the P450 superfamily, while many bacteria contain enzymes related to the Pseudomonas putida GPol membrane-bound alkane hydroxylase system. Short-chain alkanes are probably oxidized by alkane hydroxylases related to the soluble and particulate methane monooxygenases. Only the membrane-bound enzymes have been studied with respect to their prevalence in environments such as soils or aquifers.

32) Spain JC; Nishino SF; Witholt B; Tan LS; Duetz WA (2003)

Production of 6-phenylacetylene picolinic acid from diphenylacetylene by a toluene-degrading Acinetobacter strain

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69: 4037-4042

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a How rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.

31) Duetz WA; Bouwmeester H; van Beilen JB; Witholt B (2003)

Biotransformation of limonene by bacteria, fungi, yeasts, and plants

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 61: 269-277

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.

30) van Beilen JB; Duetz WA; Schmid A; Witholt B (2003)

Practical issues in the application of oxygenases

TRENDS IN BIOTECHNOLOGY 21: 170-177

Oxygenases carry out the regio-, stereo- and chemoselective introduction of oxygen in a tremendous range of organic molecules. This versatility has already been exploited in several commercial processes. There are, however, many hurdles to further practical large-scale applications. Here, we review various issues in biocatalysis using these enzymes, such as screening strategies, overoxidation, uncoupling, substrate uptake, substrate toxicity, and oxygen mass transfer. By addressing these issues in a systematic way, the productivity of promising laboratory scale biotransformations involving oxygenases may be improved to levels that allow industry to realise the full commercial potential of these enzymes.

29) O'Leary ND; Duetz WA; Dobson ADW; O'Connor KE (2002)

Induction and repression of the sty operon in Pseudomonas putida CA-3 during growth on phenylacetic acid under organic and inorganic nutrient-limiting continuous culture conditions

FEMS MICROBIOLOGY LETTERS 208: 263-268

The effects of various nutrient-limitin conditions on expression of the sty operon in Pseudomonas putida CA-3 were investigated. It was observed that limiting concentrations of the carbon source phenylacetic acid, resulted in high levels of phenylacetyl coenzyme A (CoA) ligase activity, this was accompanied also by upper pathway styrene monooxygenase enzyme activity. The introduction of inorganic nutrient limitations, (nitrate, sulfate and phosphate), caused a dramatic reduction in detectable levels of phenylacetyl CoA ligase activity, particularly in the presence of the primary carbon source, succinate. Under these conditions it was no longer possible to detect styrene monooxygenase activity. Reverse transcription PCR analysis of total RNA, isolated under each of the continuous culture conditions examined, revealed that variations in the levels of enzyme activity coincided with altered patterns of corresponding paaK (phenylacetyl CoA ligase) and styA (styrene monooxygenase) gene expression. Transcription of the upper pathway regulatory sensor kinase gene styS was also observed to be growth condition-dependent. These observations suggest that induction/repression of the sty operon in P. putida CA-3, during growth on phenylacetic acid under continuous culture conditions, involves regulatory mechanisms coordinately affecting both the upper and lower pathways and acting at the level of gene transcription. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

28) Li Z; van Beilen JB; Duetz WA; Schmid A; de Raadt A; Griengl H; Witholt B

Oxidative biotransformations using oxygenases

CURRENT OPINION IN CHEMICAL BIOLOGY 2002, Vol 6, Iss 2, pp 136-144

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.

27) Li Z; Feiten HJ; Chang DL; Duetz WA; van Beilen JB; Witholt B

Preparation of (R)- and (S)-N-protected 3-hydroxypyrrolidines by hydroxylation with Sphingomonas sp HXN-200, a highly active, regio- and stereoselective, and easy to handle biocatalyst

JOURNAL OF ORGANIC CHEMISTRY 2001, Vol 66, Iss 25, pp 8424-8430

Hydroxylation of N-benzylpyrrolidine 8 with resting cells of Sphingomonas sp. HXN-200 gave N-benzyl-3-hydroxypyrrolidine 15 in 53% ee (S) with an activity of 5.8 U/g CDW. By changing the "docking/protecting group" in pyrrolidines, hydroxylation activity and enantioselectivity were further improved and the enantiocomplementary formation of 3-hydroxypyrrolidines was achieved: hydroxylation of N-benzoyl-, N-benzyloxycarbonyl-, N-phenoxycarbonyl-, and N-tert-butoxycarbonyl-pyrrolidines 9-12 gave the corresponding 3-hydroxypyrrolidines 16-19 in ee of 52% (R), 75% (R), 39% (S), and 23% (R), respectively, with an activity of 2.2, 16, 14, and 24 U/g CDW, respectively. Simple crystallizations increased the ee of 16-18 to 95% (R), 98% (R), and 96% (S), respectively. Hydroxylation of pyrrolidines 8-12 with soluble cell-free extracts of Sphingomonas sp. HXN-200 and equimolar NADH gave 3-hydroxypyrrolidines 15-19 in nearly the same ee as the products generated by whole cell transformation, suggesting that this strain possesses a novel soluble alkane monooxygenase. Cells of Sphingomonas sp. HXN-200 were produced in large amounts and could be stored at -80 degreesC for 2 years without significant loss of activity. The frozen cells can be thawed and resuspended for biohydroxylation, providing a highly active and easy to handle biocatalyst for the regio- and stereoselective hydroxylation of nonactivated carbon atoms. These cells were used to prepare 1.0-3.2 g (66.4-93.5% yield) of 3-hydroxypyrrolidines 16-19 by hydroxylation of pyrrolidines 9-12 on 0.9-2 L scale. Preparative hydroxylation was also achieved with growing cells as biocatalysts; hydroxylation of pyrrolidine 11 on 1 L scale gave 1.970 g (79.7% yield) of 3-hydroxypyrrolidine 18.

26) Duetz WA; van Beilen JB; Witholt B

Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis

CURRENT OPINION IN BIOTECHNOLOGY 2001, Vol 12, Iss 4, pp 419-425

The unique catalytic properties of oxygenases (the regio-specific and/or enantio-specific hydroxylation of non-activated carbons) are of undisputed biosynthetic value. Factors that govern the economics of their industrial use include a low k(cat), a frequently decreased k(cat) in recombinant strains, limiting oxygen transfer rates in bioreactors, product inhibition, and the demanding discovery (screening) process.

25) Duetz WA; Fjallman AHM; Ren SY; Jourdat C; Witholt B

Biotransformation of D-limonene to (+) trans-carveol by toluene-grown Rhodococcus opacus PWD4 cells

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2001, Vol 67, Iss 6, pp 2829-2832

The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate D-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])(-1), and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the D-limonene conversion. Glucose-grown cells did not form any trans-carveol from D-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.

24) O'Leary ND; O'Connor KE; Duetz W; Dobson ADW

Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3

MICROBIOLOGY-SGM 2001, Vol 147, pp 973-979

The styrene degradative pathway in Pseudmonas putida CA-3 has previously been shown to be divided into an upper pathway involving the conversion of styrene to phenylacetic acid and a lower pathway for the subsequent degradation of phenylacetic acid. It is reported here that expression of the regulatory genes styS and styR is essential for transcription of the upper pathway, but not for degradation of the lower pathway inducer, phenylacetic acid. The presence of phenylacetic acid in the growth medium completely repressed the upper pathway enzymes even in the presence of styrene, the upper pathway inducer. This repression is mediated at the transcription level by preventing expression of the styS and styR regulatory genes. Finally. an examination was made of the various stages of the diauxic growth curve obtained when P. putida CA-3 was grown on styrene together with an additional carbon source and it is reported that catabolite repression may involve a different mechanism to transcriptional repression by an additional carbon source.

23) Minas W; Bailey JE; Duetz W

Streptomycetes in micro-cultures: Growth, production of secondary metabolites, and storage and retrieval in the 96-well format

ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY 2000, Vol 78, Iss 3-4, pp 297-305

Mycelium-forming Streptomyces strains were grown in one milliliter liquid micro-cultures in square deep-well microtiter plates. Growth was evaluated with respect to biomass formation and production of secondary metabolites which were found to be very similar in the micro-cultures, bioreactor, and shake flask cultivations, respectively. Despite repetitive sampling and extensive growth on the walls of the wells, no cross contamination occurred. Furthermore, we successfully employed cold storage at -20 degreesC of spore suspensions (in the 96-well format), directly prepared from cultures grown on agar in the microtitre plate. Cultures were retrieved by replicating aliquots from the frozen spore suspensions.

22) Duetz WA; Witholt B

Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates

BIOCHEMICAL ENGINEERING JOURNAL 2001, Vol 7, Iss 2, pp 113-115

Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5 cm at 300 rpm (24 mmol O-2 l(-1) h(-1) at a culture volume of 0.75 ml) in comparison to a shaking diameter of 2.5 cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5 cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5 cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow. (C) 2001 Elsevier Science B.V. All rights reserved.

21) Duetz, W.A., Minas, W., Kuhner, M., Clerval, R., Fjallman, A.H.M., Witholt, B. (2001). Miniaturized microbial growth systems in screening. Bioworld 2: 8-10

20) O'Connor KE, Witholt B, Duetz W (2001).

p-Hydroxyphenylacetic acid metabolism in Pseudomonas putida F6

JOURNAL OF BACTERIOLOGY 183 (3): 928-9331

Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihgdroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3, 4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage

19) Duetz WA; Ruedi L; Hermann R; O'Connor K; Buchs J; Witholt B

Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2000, Vol 66, Iss 6, pp 2641-2646

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable, We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite, Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

18) Clerval, R., Kühner, M., Li, Z., Minas, W., Fjällman, A., Witholt, B. and Duetz, W.A. (2000) The come-back of high-throughput screening of wild-type microbial strains through the use of miniaturised growth systems and LC-MS. Bioworld 6: 24-26

17) Li Z; Feiten HJ; van Beilen JB; Duetz W; Witholt B

Preparation of optically active N-benzyl-3-hydroxypyrrolidine by enzymatic hydroxylation

TETRAHEDRON-ASYMMETRY 1999, Vol 10, Iss 7, pp 1323-1333

Hydroxylation of N-benzylpyrrolidine 2 with Pseudomonas oleovorans GPol afforded 62% of (R)-N-benzyl-3-hydroxypyrrolidine 3 in 52% e.e. This reaction was catalyzed by the alkane hydroxylase system in this strain, which was demonstrated by hydroxylation of 2 with Escherichia coli GEc137 (pGEc47), a recombinant strain that carries the genes for the alkane hydroxylase system of P. oleovorans GPol. In a set of 70 alkane-degrading microorganisms, 12 were found to catalyze the biotransformation of 2 into 3 by screening with a microtiter plate technique. Hydroxylation of 2 with isolates HXN-1100 and HXN-200 gave 67% of (R)-3 in 70% e.e. and 62% of (S)-3 in 53% e.e., respectively.

16) Duetz WA; Wind B; van Andel JG; Barnes MR; Williams PA; Rutgers M

Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWW0)

MICROBIOLOGY-UK 1998, Vol 144, pp 1669-1675

Pseudomonas putida mt-2, harbouring TOL plasmid pWWO, is capable of degrading toluene and a range of di-and tri-alkylbenzenes. In this study, chemostat-grown cells (D = 0.05 h(-1), toluene or m-xylene limitation) of this strain were used to assess the kinetics of the degradation of toluene, m-xylene, p-xylene, and a number of their pathway intermediates. The conversion kinetics for the three hydrocarbons showed significant differences: the maximal conversion rates were rather similar [11-14 mmol h(-1) (g dry wt)(-1)] but the specific affinity (the slope of the v vs s curve near the origin) of the cells for toluene [1300 \ (g dry wt)(-1) h(-1)] was only 5% and 14% of those found for m-xylene and p-xylene, respectively. Consumption kinetics of mixtures of the hydrocarbons confirmed that xylenes are strongly preferred over toluene at low substrate concentrations, The maximum flux rates of pathway intermediates through the various steps of the TOL pathway as far as ring cleavage were also determined. Supply of 0.5 mM 3-methylbenzyl alcohol or 3-methylbenzaldehyde to fully induced cells led to the transient accumulation of 3-methylbenzoate. Accumulation of the corresponding carboxylic acid (benzoate) was also observed after pulses of benzyl alcohol and benzaldehyde, which are intermediates in toluene catabolism. Analysis of consumption and accumulation rates for the various intermediates showed that the maximal rates at which the initial monooxygenation step and the conversion of the carboxylic acids by toluate 1,2-dioxygenase may occur are two-to threefold lower than those measured for the two intermediate dehydrogenation steps.

15) Rutgers M; van Bommel S; Breure AM; van Andel JG; Duetz WA

Effect of pH on the toxicity and biodegradation of pentachlorophenol by Sphingomonas sp. strain P5 in nutristat culture

ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 1998, Vol 17, Iss 5, pp 792-797

A polychlorinated-phenol degrading bacterium, Sphingomonas sp. strain P5, was grown in nutristat culture (i.e., a continuous culture at a controlled substrate concentration) with pentachlorophenol (PCP) as the sole carbon and energy source. During steady state conditions, the effect of the medium pH on the growth of strain P5 on PCP was established. At lower pH values PCP exhibited a stronger toxicity than at higher pH values. Inhibition of the growth of strain P5 by PCP was correlated to the concentration of the undissociated phenol in the system, rather than to the dissociated or total PCP concentration. The results indicate that acidification of natural environments may enhance the toxicity of chlorophenols and suggest that treatments to increase environmental pH may reduce risk of chlorophenol toxicity at acidified sites.

14) Rutgers M; Breure AM; vanAndel JG; Duetz WA

Growth yield coefficients of Sphingomonas sp. strain P5 on various chlorophenols in chemostat culture

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 1997, Vol 48, Iss 5, pp 656-661

A polychlorophenol-degrading bacterium, Sphingomonas sp. strain P5, was grown in 2,6-dichlorophenol(26-DCP)-limited. 2,3,6-trichlorophenol(236-TCP)-limited, 2,4,6-trichlorophenol(236-TCP)-limited, 2,3,4,6-tetrachlorophenol(2346-TeCP)-limited, and pentachlorophenol(PCP)-limited chemostat cultures at a dilution rate of 0.02 +/- 0.002 h(-1). The cultures were analyzed for the yield coefficient for growth on chlorophenol during steady-state conditions. The average growth yields coefficients (as carbon conversion efficiencies) were 0.252, 0.230, 0.219, 0.157, and 0.121 mol C mol C-1 for 26-DCP, 236-TCP, 246-TCP, 2346-TeCP, and PCP respectively. The differences in growth yield can be interpreted in terms of the energetics of chlorinated carbon metabolism; i.e. substitution of the phenol moiety reduces the available metabolic energy by one electron per chlorine. The growth yield coefficients on chlorinated phenols were lower than the yield coefficients of heterotrophic growth reported in the literature on non-chlorinated and aliphatic compounds. Metabolic origins for low growth yield coefficients on (chlorinated) aromatic compounds are postulated.

13) Filonov AE; Duetz WA; Karpov AV; Gaiazov RR; Kosheleva IA; Breure AM; Filonova IF; vanAndel JG; Boronin AM

Competition of plasmid-hearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 1997, Vol 48, Iss 4, pp 493-498

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D = 0.05 h(-1)) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive.

12) Barnes MR; Duetz WA; Williams PA

A 3-(3-hydroxyphenyl)propionic acid catabolic pathway in Rhodococcus globerulus PWD1: Cloning and characterization of the hpp operon

JOURNAL OF BACTERIOLOGY 1997, Vol 179, Iss 19, pp 6145-6153

Rhodococcus globerulus PWD1, a soil isolate from a polluted site in The Netherlands, is able to degrade a broad range of aromatic compounds, A novel gene cluster which appears to encode a pathway for the degradation of phenolic acids such as 3-(3-hydroxyphenyl) propionate (3HPP) has been cloned from the chromosome of this organism, Sequence analysis of a 7-kb region identified five open reading frames (ORFs), Analysis of mRNA showed that the genes were expressed during growth on 3HPP and 3-hydroxyphenylacetate (3HPA) but not during growth on m-cresol or succinate, The first ORF, hppA, which appears to be separately transcribed, had considerable amino acid identity with a number of hydroxylases. Transcriptional analysis indicates that the next four ORFs, hppCBKR, which are tightly clustered, constitute a single operon, These genes appear to encode a hydroxymuconic semialdehyde hydrolase (HppC), an extradiol dioxygenase (HppB), a membrane transport protein (HppK), and a member of the IcIR family of regulatory proteins (HppR), The activities of HppB and HppC have been confirmed by enzyme assay of Escherichia coli hosts, The substrate specificity of HppB expressed from the cloned gene matches that of the meta-cleavage dioxygenase expressed from wild-type Rhodococcus grown on both 3HPP and 3HPA and is considerably more active against acid than against neutral catechols, The deduced amino acid sequences of the gene products have a recognizable homology with a broad range of enzymes and proteins involved in biodegradation and appear most similar to the mhp operon from E. coli K-12, which also encodes the degradation of 3HPP.

11) Duetz WA; Wind B; Kamp M; vanAndel JG

Effect of growth rate, nutrient limitation and succinate on expression of TOL pathway enzymes in response to m-xylene in chemostat cultures of Pseudomonas putida (pWW0)

MICROBIOLOGY-UK 1997, Vol 143, pp 2331-2338

Previous studies have shown that expression of the toluene and m- and p-xylene degradation pathway in Pseudomonas putida (pWWO) is subject to catabolite repression by succinate. We report here that the expression level of the upper part of this so-called TOL pathway in cells grown in chemostat culture is strongly influenced by nutrient limitation when m-xylene is the sole carbon and energy source. The benzylalcohol dehydrogenase (BADH) levels in cells that are growth-limited by anabolic processes [sulphate (S)-, phosphate (P)- or nitrogen (N)-limiting conditions] were 3-12% of those in cells growing under oxygen limitation (when catabolism limits growth). BADH levels under S-, P- and N-limitation were further decreased (three- to fivefold) when succinate was supplied in addition to m-xylene. Levels of the meta-cleavage pathway enzyme catechol 2,3-dioxygenase were less affected by the growth conditions but the general pattern was similar. Dilution rate also influenced the expression of the TOL pathway: BADH levels gradually decreased with increasing dilution rates, from 1250 mU (mg protein)(-1) at D = 0.05 h(-1) under m-xylene limitation to 290 mU (mg protein)(-1) at D = 0.58 h(-1) (non-limited growth). BADH levels were shown to be proportional to the specific affinity of whole cells for m-xylene. It may, therefore, be expected that natural degradation rates are adversely affected by anabolic nutrient limitations, especially at relatively low concentrations of the xenobiotic compound.

10) OConnor K; Duetz W; Wind B; Dobson ADW

The effect of nutrient limitation on styrene metabolism in Pseudomonas putida CA-3

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1996, Vol 62, Iss 10, pp 3594-3599

Styrene degradation in Pseudomonas putida CA-3 has previously been shown to be subject to catabolite repression in batch culture. We report here on the catabolite-repressing effects of succinate and glutamate and the effects of a limiting inorganic-nutrient concentration on the styrene degradation pathway of P. putida CA-3 in a chemostat culture at low growth rates (0.05 h(-1)). Oxidation of styrene and the presence of styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities were used as a measure of the expression of the styrene degradation pathway. Both glutamate and succinate failed to repress the styrene degradation ability under growth conditions of carbon and energy limitation. Lower levels of enzyme activities of the styrene degradation pathway were seen in cells grown on styrene or phenylacetic acid (PAA) under conditions of both ammonia and sulfate limitation than were seen under carbon and energy limitation. Cells grown on PAA under continuous culture oxidize styrene and styrene oxide and possess styrene oxide isomerase and NAD(+)-dependent phenylacetaldehyde dehydrogenase activities. Catabolite repression of styrene metabolism was observed in cells grown on styrene or PAA in the presence of growth-saturating (nonlimiting) concentrations of succinate or glutamate under sulfate limitation.

9) Duetz WA; Marques S; Wind B; Ramos JL; vanAndel JG

Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWWO under various conditions of nutrient limitation in chemostat culture

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1996, Vol 62, Iss 2, pp 601-606

In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWWO) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer, This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h(-1)) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway, When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source.

8) DUETZ WA; DEJONG C; WILLIAMS PA; VANANDEL JG

COMPETITION IN CHEMOSTAT CULTURE BETWEEN PSEUDOMONAS STRAINS THAT USE DIFFERENT PATHWAYS FOR THE DEGRADATION OF TOLUENE

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1994, Vol 60, Iss 8, pp 2858-2863

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h(-1)), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxgygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus aas also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.

7) DUETZ WA; MARQUES S; DEJONG C; RAMOS JL; VANANDEL JG

INDUCIBILITY OF THE TOL CATABOLIC PATHWAY IN PSEUDOMONAS-PUTIDA (PWW0) GROWING ON SUCCINATE IN CONTINUOUS-CULTURE - EVIDENCE OF CARBON CATABOLITE REPRESSION CONTROL

JOURNAL OF BACTERIOLOGY 1994, Vol 176, Iss 8, pp 2354-2361

The TOL catabolic genes in Pseudomonas putida(pWW0) are clustered in the upper operon, encoding enzymes for the conversion of toluene and xylenes to benzoate and toluates, and the meta-cleavage operon, encoding enzymes for the conversion of the benzoate and toluates to tricarboxylic acid cycle intermediates. In this study, it was shown that cells growing in a chemostat under succinate growth-limiting conditions express both the upper and meta-cleavage pathways in response to o-xylene, a nonmetabolizable effector of the XylR regulatory protein. The dilution rate maintained in the succinate-limited chemostat cultures influenced the synthesis levels of TOL pathway enzymes, their steady-state levels, and their turnover rates. Cells growing in the presence of nonlimiting concentrations of succinate in continuous culture did not express pathway enzymes in response to the addition of o-xylene, which was due to a blockage at the transcriptional level. Expression of the meta-cleavage pathway in response to 2,3-dimethylbenzoate, a nonmetabolizable effector of the XylS regulatory protein, was 93% lower in cultures exposed to succinate at nonlimiting concentrations than in the succinate-limited chemostats. The mRNA level of xylS during nonlimited growth on succinate was very low compared with that in succinate-limited cultures, suggesting that suppression of expression of the meta-cleavage pathway is regulated mainly by the level of the XylS regulator.

6) LLOYDJONES G; DEJONG C; OGDEN RC; DUETZ WA; WILLIAMS PA

RECOMBINATION OF THE BPH (BIPHENYL) CATABOLIC GENES FROM PLASMID PWW100 AND THEIR DELETION DURING GROWTH ON BENZOATE

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1994, Vol 60, Iss 2, pp 691-696

Pseudomonas sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph(+) phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of bph deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the bph genes on the wild-type plasmid and the RP4 cointegrate plasmid.

5) DUETZ WA; WINSON MK; VANANDEL JG; WILLIAMS PA

MATHEMATICAL-ANALYSIS OF CATABOLIC FUNCTION LOSS IN A POPULATION OF PSEUDOMONAS-PUTIDA MT-2 DURING NON-LIMITED GROWTH ON BENZOATE

JOURNAL OF GENERAL MICROBIOLOGY 1991, Vol 137, pp 1363-1368

Pseudomonas putida mt-2, harbouring the TOL plasmid PWW0, was grown continuously on benzoate in a phauxostat at a non-limited rate. The gradual decrease in the population carrying the complete TOL plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (TOL- cells). The growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h-1; v = 0.11 h-1) and by mathematical analysis of the ingrowth of TOL- cells (v = 0.12 h-1). The latter procedure also established that the segregation rate was of the order of magnitude 10(-5) h-1. Similar values for the growth-rate advantage and the segregation rate were found when both benzoate and succinate were present in non-limiting concentrations. It is suggested that the growth-rate disadvantage of the wild-type strain is caused by inhibitory effects of an intermediate in the degradation of benzoate via the plasmid-encoded meta-pathway.

4) DUETZ WA; VANANDEL JG

STABILITY OF TOL PLASMID PWW0 IN PSEUDOMONAS-PUTIDA MT-2 UNDER NONSELECTIVE CONDITIONS IN CONTINUOUS CULTURE

JOURNAL OF GENERAL MICROBIOLOGY 1991, Vol 137, pp 1369-1374

Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and zylene (TOL- cells), was determined. Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1). In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.

3) Breure, A.M., Duetz, W.A., Zoutberg, G.R., Mulder, R., Andel, J.G. van (1986) Measurement of the rate of hydrogen production by Clostridium butyricum using an amperometric method. FEMS Microbiol. Lett. 33, 289‑292

2) De Vries, G.J., Duetz, W., Buijs, R.M., van-Heerikhuize, J., and Vreeburg, J.T. (1986) Effects of androgens and estrogens on the vasopressin and oxytocin innervation of the adult rat brain. Brain Res. 10: 399: 296-302

1) Van der Bend, R.L., Duetz W., Colen, A.M., Van Dam,K., Berden J.A. (1985) Differential effects of triphenyltin and 8-azido-ATP on the ATP synthesis, ATP-Pi exchange, and ATP hydrolysis in liposomes containing ATP synthase and bacteriorhodopsin. Arch. Biochem. Biophys. 241: 461-71

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