Environmental Microbiology (1999) 1(2), 167–174
Towards elucidation of microbial community metabolic pathways: unravelling the network of carbon sharing in a pollutant-degrading bacterial consortium by immunocapture and isotopic ratio mass spectrometry Oliver Pelz,† Michael Tesar, Rolf-Michael Wittich, Edward R. B. Moore, Kenneth N. Timmis and WolfRainer Abraham* Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Summary Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4chlorosalicylate as carbon substrate within a simple chemostat microbial community using 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites. Introduction The activities of microorganisms have been studied traditionally in monocultures, and important progress in the elucidation of individual metabolic pathways and underlying mechanisms has been gained thereby. However, in nature, microorganisms live in mixed communities of various complexities that are generally characterized by considerable metabolic and phylogenetic diversity. Little is Received 13 September, 1998; revised 4 December, 1998; accepted 17 December, 1998. †Present address: Swiss Federal Institute of Technology (ETH Zu¨rich), Institute of Terrestrial Ecology, Soil Biology, Grabenstrasse 3, CH-8952 Schlieren, Switzerland. *For correspondence. E-mail
[email protected]; Tel. (þ49) 531 6181 419; Fax (þ49) 531 6181 411. 䊚 1999 Blackwell Science Ltd
known about how microbial communities function as biological units, how their activities are regulated by ecological interactions between the community members or about the metabolic routes that are followed by available nutritional resources within the community as an entity. If we are to understand microbial control of environmental quality, the role of microbes in global change, the ecological parameters regulating algal blooms, etc., we must extend our understanding of the cellular metabolism of individual organisms to that of the community as a biological entity, analyse the sharing of resources and the functional roles of the different members and characterize the principal parameters and interactions that regulate the activities of the community. A major hindrance to such investigations is the paucity of adequate methods. However, the increasing availability of stable isotope-labelled nutrients and the exceptional sensitivity of measurement of isotope enrichment in cell materials by isotopic ratio mass spectrometry, coupled with the growing number of taxon-specific ‘biomarkers’ being identified, begins to provide the experimental opportunity for investigating community metabolic routes and the critical ecological interactions that determine and influence them. We report here an analysis of carbon sharing in a carbon-limited chemostat community growing on 4chlorosalicylate, an intermediate in the aerobic degradation of important organic environmental pollutants such as 3-chlorodibenzofuran (Harm et al., 1991) and 2-chloronaphthalene (Morris and Barnsley, 1982). This study has exposed an intricate network of carbon sharing in the community, defined the ecological roles of its three dominant members and revealed that the substrate is catabolized by two completely different parallel routes, one of which is novel and involves protoanemonin as a critical intermediate, a toxic substance not previously found as a pathway intermediate in the microbial world (Blasco et al., 1995; 1997).
Results The experimental microbial community studied here was grown as an aerated carbon-limited continuous culture in a phosphate-buffered mineral salts medium containing
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Fig. 1. Effects of increasing dilution rate in chemostats of the community and of Pseudomonas sp. strain MT1. The initial dilution rate of 0.16 day¹1 was increased to 0.2 day¹1 (both chemostats), 0.24 day¹1, 0.32 day¹1, 0.4 day¹1, 0.48 day¹1, 0.64 day¹1 and 0.8 day¹1 (community chemostat). The chemostat outflows were monitored for microbial densities (OD600), viable bacteria (plate counting) (A, community chemostat; C, MT1 chemostat) and concentrations of substrate and metabolites (B, community; D, MT1 chemostat).
5 mM 4-chlorosalicylate (4CS) (Faude, 1995). The inoculum for the chemostat was taken from the aerobic zone of the sediment of the Spittelwasser, a small tributary of the Elbe River system near Bitterfeld, Sachsen-Anhalt, Germany, which served for decades for the disposal of untreated waste water from the chemical industries concentrated in this region. Until the recent shutdown of most of these industries, the Spittelwasser contained no eukaryotes, but a rich diversity of prokaryotes (Mau and Timmis, 1998). Once the microbial community in the chemostat reached equilibrium, it consisted principally of four organisms, which were identified by determination of their 16S rRNA gene sequences as two different Pseudomonas spp. (MT1 and MT4), an Alcaligenes sp. (MT3) and an Empedobacter sp. (MT2). Polyclonal rabbit antisera specific for each of the members of the community were raised and used to measure the relative abundances in the chemostat. These were 84 ⫾ 3% for MT1, 8 ⫾ 4% for MT3 and MT4 and 1% for MT2. This community composition has been stably maintained over a period of more than 3 years. ‘Daughter’ chemostats have been subjected to a
variety of perturbations, during which the composition of the community was observed to change substantially, but after which it eventually returned to that of the ‘parent’ chemostat (A. Rabenau, unpublished). As can be seen in Fig. 1, the consortium tolerated a stepwise increase in dilution rate from 0.16 day¹1 to 0.64 day¹1; at such dilution rates, all substrate was consumed, and no secreted metabolites were observed. However, when the dilution rate was increased to 0.8 day¹1, the toxic product protoanemonin (Blasco et al., 1997) accumulated in and poisoned the chemostat. Characterization of the catabolic potential of the individual members of the community revealed that only MT1 is able to transform and grow with the substrate 4CS as the sole source of carbon and energy. This explains its high abundance in the chemostat. However, in batch culture, it only tolerated substrate concentrations as high as 1 mM, above which it did not survive. Cultivation of MT1 as a monoculture in an identical parallel chemostat revealed that it could grow at the low dilution rate initially selected, but it is very sensitive to small changes, and it 䊚 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 167–174
Network of carbon sharing in a bacterial consortium 169 Table 1. Enzymatic activities in extracts of the consortium and of Pseudomonas sp. MT1.
Enzyme
Substrate
Consortium (U g¹1 protein)
Pseudomonas sp. MT1 (U g¹1 protein)
Catechol 2,3-dioxygenase (Chloro-)catechol 1,2-dioxygenase
Catechol Catechol 4-Chlorocatechol Maleylacetate; NADH
2 2104 947 4052
1 779 181 30
Maleylacetate reductase
could not tolerate an increase in dilution rate. After inoculation and during equilibration of the chemostat, 4chlorocatechol appeared transiently in the medium. Moreover, once equilibrium at the initial dilution rate of 0.16 day¹1 was reached, a further metabolite, protoanemonin, was observed. An increase in the dilution rate of only 25%, to 0.2 day¹1, resulted in the accumulation of 3-chloro-cis, cis-muconate to a level of 4.5 M and of protoanemonin to 43 M, at which point bacterial growth stopped and washout occurred. Chlorocatechols are ordinarily channelled into productive chlorocatechol (type II ortho-cleavage) pathways (Blasco et al., 1997). In the absence of such a pathway, they may be partially catabolized by catechol (type I ortho-cleavage) pathways to the dead-end intermediate protoanemonin (Blasco et al., 1995). These two pathways have been studied intensively and are readily distinguished by the substrate preferences of certain enzymes and the presence or absence of others. In order to gain information on the degradation route of 4CS in the community, enzyme assays were carried out on extracts prepared from biomass obtained from the two chemostats. No catechol-2,3-dioxygenase activity, the key enzyme of the meta-cleavage pathway (Gibson and Subramanian, 1984), was detected in either extract. As can be seen in Table 1, both extracts contain (chloro-) catechol-1,2-dioxygenase activity. In the case of the extract from the consortium, high activities were measured for both catechol and 4-chlorocatechol, whereas in the case of the MT1 extract, a low activity was measured for 4-chlorocatechol. Given the observation that MT1 comprises as much as 84% of the total community, the high activity for 4-chlorocatechol in the community extract, which presumably comes from the other members, is particularly impressive. The low ortho-cleavage activity for 4chlorocatechol of MT1 suggests that this organism may not have a chlorocatechol pathway, a suggestion confirmed by the finding that MT1 extracts contained no maleylacetate reductase activity, an enzyme characteristic of type II pathways (Blasco et al., 1995). In contrast, the consortium extract exhibited high maleylacetate reductase activity. Thus, it seems that the community exploits two different pathways for the degradation of 4CS, namely a type II chlorocatechol pathway and another, probably a type I, pathway. If this latter pathway 䊚 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 167–174
were used, and it is known that protoanemonin can be formed from 4-chlorocatechol via type I pathways (Blasco et al., 1995), then protoanemonin may be a productive intermediate in the MT1 pathway, rather than a deadend product, at least when 4CS substrate concentrations are low enough to avoid its accumulation at significant levels. This possibility was tested by offering MT1 [U-13C]labelled protoanemonin and determining whether it is metabolized and the labelled carbon atoms incorporated into phospholipids of cellular material. Labelled protoanemonin was prepared by transformation of commercially available [U-13C]-4-chlorocatechol by a cell extract of MT1 (Blasco et al., 1995), purified and added as a pulse to the MT1 chemostat. The initial concentration in the chemostat, 50 nM, is extremely low [1000-fold lower than the minimum inhibitory concentration (MIC) for MT1 (Blasco et al., 1995)] and has no observable effect on the bacteria. Phospholipids were subsequently isolated from biomass collected from the community chemostat, and the ␦13C-values of their fatty acids were measured by means of a gas chromatograph coupled via a combustion interface to an isotopic ratio mass spectrometer (GCC-IRMS; Abrajano et al., 1994). Phospholipid fatty acids, particularly the C16:0 and C16:1, were found to be significantly enriched in 13C with a ⌬␦13C þ 3 ⫾ 0.5 enrichment in C16:1 fatty acids compared with those from a control chemostat fed with the same amount of non-labelled protoanemonin. Protoanemonin can thus be taken up, channelled into central metabolic pathways and metabolized to cellular components. This supports the possibility that protoanemonin may serve as a productive metabolite in 4CS degradation. Incubation of resting cells of MT1 with 1 mM 4chlorocatechol in sodium phosphate buffer resulted in the formation of 25 M protoanemonin, 17 M 3-chlorocis,cis-muconate and 16 M cis-acetylacrylate. It has been demonstrated recently that protoanemonin can be transformed, albeit inefficiently, by the dienelactone hydrolase of Pseudomonas sp. B13 to cis-acetylacrylate (Bru¨ckmann et al., 1998). We therefore tested whether cis-acetylacrylate can serve as a sole source of carbon and energy for MT1 grown in minimal medium. This was found to be the case. Resting cells of salicylate-grown MT1 cells metabolized 1 mM cis-acetylacrylate within
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24 h. These results confirm that protoanemonin and cisacetylacrylate can serve as carbon sources for MT1 and strongly suggest that 4CS is metabolized via a novel pathway with 4-chlorocatechol, 3-chloro-cis,cis-muconate, protoanemonin and cis-acetylacrylate as intermediates. Given that MT1 can productively metabolize 4CS via a catechol pathway involving protoanemonin as intermediate, the question arose as to what roles the other members of the consortium play and why the consortium is more robust when confronted with higher substrate concentrations. We therefore analysed the fate of the major intermediates released by MT1, namely 4-chlorocatechol and protoanemonin. In order to do this, we introduced [U-13C]-4-chlorocatechol or -protoanemonin as pulses into the consortium chemostat and at intervals thereafter collected samples. To determine the contribution of the individual strains to the degradation of these intermediates, we used the strain-specific antibodies (Faude, 1995) to separate the consortium into its component populations by immunocapture. The level of 13C-enrichment in the bacteria-specific (White et al., 1979) C16:1 fatty acids of the phospholipids extracted from the individual populations was then determined by isotopic ratio mass spectrometry. Pulse feeding of the consortium with 0.6 M [U-13C]-4-chlorocatechol resulted in an overall 13C enrichment in the consortium of ⌬␦13C þ 24 at 6 h. The highest 13 C enrichment of ⌬␦13C þ 1557 was found for Alcaligenes sp. MT3 at 2.5 h, while that for Pseudomonas sp. MT1 was only þ 126 at 6 h and that for Pseudomonas sp. MT4 was þ 24 at 6 h (Fig. 2A). These measurements demonstrate that MT3 has an exceptionally high affinity for 4chlorocatechol and very effectively scavenges essentially all the 4-chlorocatechol released by MT1. The enzyme activities reported above are consistent with the assumption that MT3 metabolizes 4-chlorocatechol via a chlorocatechol pathway. When 1.8 M [U-13C]-protoanemonin was pulsed into the chemostat, the 13C enrichment in the total community was 45.7 at 6 h. The 13C enrichment in C16:1 of Pseudomonas sp. MT4 was ⌬␦13C 17.6 at 2.5 h and considerably lower, ⌬␦13C 3.0–4.64, for fatty acids of strains MT1 and MT3 at 2.5 h and 6 h, although these values rose to ⌬␦13C 14.9 (MT1) and 10.8 (MT3) by 24 h (Fig. 2B). The rapid and substantial 13C enrichment in the C16:1 of MT4 demonstrated a high affinity of this organism for protoanemonin and indicated that MT4 effectively scavenges most of this metabolite as it is released by MT1. The slow enrichment of 13C in MT1 and MT3 might result from the direct but slow uptake of residual protoanemonin from the pulse or, more likely, from the uptake of 13C-labelled metabolites of protoanemonin, such as cis-acetylacrylate, released by MT4. Owing to the low abundance of Empedobacter sp. MT2, it was not possible to determine the isotopic ratios of fatty
acids from immunocaptured cells of this community member. However, analysis of the individual strains of the consortium in pure culture, using unlabelled yeast extract as a carbon source and 0.2 mM chlorocatechol, partially enriched with [U-13C]-4-chlorocatechol to a ␦13C of 500, revealed assimilation of 4-chlorocatechol into the biomass of MT1, MT3 and MT4, but not MT2 (Pelz et al., 1997). This result and the low abundance of MT2 suggests that it survives on other metabolites and/or perhaps on cell debris, with no active involvement in the degradation of 4-chlorosalicylate. Discussion Figure 3 summarizes the flow of chlorosalicylate substrate carbon through the chemostat community, as revealed by the data presented here. MT1 is the only member able to metabolize 4-chlorosalicylate and constitutes the dominant population in the chemostat. A significant amount, almost 10%, of the substrate carbon spills
Fig. 2. Incorporation of stable isotope-labelled metabolites into biomarker fatty acids of individual consortium members. Aliquots of 0.6 M labelled 4-chlorocatechol (A) or 1.8 M labelled protoanemonin (B) were added as a pulse to the chemostat consortium. Samples were collected at the indicated times, the individual bacterial populations separated by immunocapture and, for each population, the isotopic ratios of bacterial biomarker phospholipid fatty acids C16:1 were determined by GC-C-IRMS. 䊚 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 167–174
Network of carbon sharing in a bacterial consortium 171 out from MT1 as the first metabolite, 4-chlorocatechol, and is very efficiently taken up by MT3 and metabolized via what is likely to be a classical chlorocatechol pathway. MT3 thereby protects MT1 from the toxicity that would otherwise accrue from a build-up of 4-chlorochatechol, a metabolite presumably reflecting a kinetic bottleneck in the MT1 pathway. MT1 also releases small amounts (in the range of 1% or less of total substrate carbon) of the next metabolite in the pathway, 3-chloro-cis,cis-muconate, which may be taken up by MT3 and/or MT4. However, it is the next metabolite in the pathway that represents a second important carbon spill by MT1 and provides a major surprise. Protoanemonin is a natural product of certain members of the Ranunculaceae family (Seegal and Holden, 1945) and has broad antibiotic activity (Didry et al., 1991). Although not a natural product found so far in microorganisms, protoanemonin has been shown to be formed during microbial metabolism of chlorinated aromatics as a toxic dead-end product that kills the producing strain (Blasco et al., 1995) and, when produced in microbial communities, is assumed to kill other members. Washout of the MT1 chemostat at an only slightly elevated dilution rate was correlated directly with the accumulation of protoanemonin, so this observation is consistent with earlier findings. The microbial community, however, is extremely stable over a long period of time over a fourfold range of dilution rates, the higher of which is presumably characterized by high levels of protoanemonin release by MT1. In this case, it is MT4 that takes up and metabolizes protoanemonin efficiently and thereby protects MT1 from suicide by poisoning. MT4 thus has a productive pathway for the metabolism of toxic protoanemonin. As (i) resting cells of MT1 form protoanemonin and cisacetylacrylate when incubated with 4-chlorocatechol; (ii) at least one dienlactone hydrolase has been shown to be able to convert protoanemonin to cis-acetylacrylate; and (iii) MT1 is able to use cis-acetylacrylate as a sole source of carbon and energy, it seems likely that the primary route of 4-chlorosalicylate metabolism in the community, namely by MT1, involves a novel pathway with toxic protoanemonin as a critical metabolite that is channelled via cisacetylacrylate by as yet unknown reactions into the Krebs cycle. This pathway probably also operates in MT4. In addition, the classical chlorocatechol pathway operates in parallel in the community, although it seems to be responsible for the metabolism of less than 10% of the total substrate carbon. Moreover, MT3 also has the capacity to catabolize either protoanemonin or a metabolite thereof productively, although quantitatively this route may not play a significant role in the metabolism of 4chlorosalicylate by the community. In conclusion, the use of 13C-labelled substrates coupled with isotopic ratio mass spectometry to measure isotopic enrichment in immunocaptured individual 䊚 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 167–174
populations of a stable microbial consortium is shown to be a powerful approach for exploring community metabolism and has revealed a highly complex system of carbon sharing involving a catabolic pathway network consisting of at least two routes, the major one of which is novel. The community seems to be so stable because each member plays a crucial role, either in providing carbon skeletons for the others (MT1) or in scavenging toxic metabolites that inhibit the primary degrader if they accumulate. Additional interdependencies, such as cross-feeding of growth factors, may well exist but were not analysed in this study. From the results presented here, it would seem that metabolic and physiological weaknesses of primary degraders of xenobiotics may be effectively compensated for by recruitment of other organisms with appropriate complementary physiology to build a consortium that, as a biological unit, is robust and able to extract the maximal metabolic benefit from the nutritional opportunity. The period since 1950 has been a golden age in the elucidation of metabolic routes within individual species. The challenge now is to elucidate metabolic networks in natural biological assemblages (and models thereof) in which metabolites produced via a pathway of one cell type flow to other cells to enter new pathways. The surprises exposed in the present study are surely only a mere taste of what is in store! Exploration of metabolic networks in natural assemblages will ultimately yield an understanding of the functioning of such assemblages as biological units, of the roles of the component members and of their metabolic, physiological and energetic benefits and sacrifices as team players, and how the assemblage as a unit interacts with its abiotic environment. Such an understanding will provide the ground rules for interventions to influence environmental processes and to optimize biotechnological applications in the environment, such as in situ bioremediation.
Experimental procedures
Chemostat conditions The microbial community studied here was grown in a 5 l vessel as an aerated 3 l carbon-limited continuous culture in a phosphate-buffered mineral salts medium (7.8 g of Na2HPO4 × 2H2O, 6.8 g of KH2PO4, 410 mg of MgSO4 × 7H2O, 10 mg of NH4-Fe-citrate, 50 mg of Ca(NO3)2 × 4H2O, 85 mg of NaNO3, 13 l of concentrated HCl, 0.7 mg of ZnCl2, 1 mg of MnCl2 × 4H2O, 0.62 mg of H3BO3, 1.9 mg of CoCl2 × 6H2O, 0.17 mg of CuCl2 × 2H2O, 0.24 mg of NiCl2 × 6H2O, 0.36 mg of NaMoO4 × 2H2O in 1 l of H2O), containing 5 mM 4-chlorosalicylate (4CS). The inoculum for the chemostat was 10 ml of sediment taken from the upper 2–3 mm aerobic zone of the Spittelwasser. The chemostat was maintained at 12⬚C, which is approximately the mean
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Network of carbon sharing in a bacterial consortium 173 temperature of the Spittelwasser sediment, at a dilution rate of 0.16 day¹1.
Enumeration of bacteria Serial dilutions were made in 0.85% NaCl. Colony-forming units (cfu) were determined from 100 l of the appropriate dilution spread on 1/10 Luria–Bertani (LB) medium (1 g l¹1 tryptone, 1 g l¹1 NaCl, 0.5 g l¹1 yeast extract) agar plates at room temperature and counted on at least two different plates after 3 days.
Generation of rabbit antisera Bacteria were grown to an OD600 of 0.3, washed three times in PBS, fixed in PBS containing 2% formaldehyde, washed again three times in PBS, aliquoted and stored at ¹70⬚C. A 1 ml aliquot corresponding to an OD600 of 0.3 was mixed with an equal volume of incomplete Freund’s adjuvant (Sigma) and the emulsion injected subcutaneously into a female rabbit. Four booster injections were given at intervals of 3 weeks, and test samples were taken at the same time. All sera were tested in immunofluorescence using all four chemostat isolates as pure cultures. Rabbit antisera that showed ‘ring-like’ surface labelling of cells were tested subsequently for cross-reactivity against other chemostat isolates.
Indirect immunofluorescence A formaldehyde-fixed chemostat sample (5 l) was diluted in PBS and filtered through a filter sandwich (0.2 m) composed of a polycarbonate filter (Costar), followed by a nitrocellulose filter support (Sartorius). Bacteria were incubated for 2 h with the rabbit antiserum diluted 1:250 in PBS supplemented with 2% fetal calf serum (FCS). After three washing steps in PBS supplemented with 0.05% Tween 20 (PBST), a Cy3-labelled sheep anti-mouse IgG antibody (Sigma) was used according to the instructions of the supplier. After the final washing step, cells were stained by SYBR stain (Molecular Probes), the polycarbonate filter was removed and mounted in glycerol supplemented with 2.5% 1,4-diazobicyclo[2,2,2]-octane (Merck) on a microscopic slide. A fluorescence-equipped microscope (Axioplan; Zeiss) was applied for cell counting, using the following filtersets (Zeiss): #09 for SYBR green staining (Molecular Probes; excitation: 450–490 nm; emission: 520 nm), #03 for Cy3 (excitation: 546 nm; emission: 590 nm).
Purification of antibodies and immunocapture Rabbit antisera (3.6 ml) were diluted 1:2 in PBS and immunoglobulins precipitated by the addition of saturated ammonium
sulphate to a final concentration of 60% (v/v; 4.5 ml). Immunoglobulins were dissolved in 4.5 ml of PBS and 1.5 ml of purified on a protein A column, according to the instructions of the supplier (Pharmacia). IgG thus purified was dialysed against PBS and analysed for purity by electrophoresis on an SDS– PAGE gel, followed by silver staining or Western blotting with a rabbit-specific peroxidase-labelled antiserum (Promega). In parallel, purified IgG was analysed for specificity by indirect immunofluorescence with various test strains. For immunocapture (Tamura et al., 1984; Chapman et al., 1997), microtitre plates (Nunc) were coated at 4⬚C overnight with 5–10 g/well of protein A-purified rabbit antibodies. Unbound antibodies were removed, and the plates were washed three times in PBS by means of an enzyme-linked immunosorbent assay (ELISA) washer and residual binding capacities blocked by a 2% solution of BSA (Sigma). After washing the plates three times in PBS, 100 l of the chemostat culture was added to each well and incubated for 2 h. Unbound cells were removed from the plates by repeated washings in PBS (six times), and bound cells were subsequently released by the addition of 150 l/well of 100 mM glycine-HCl (pH 3.0). The eluate was adjusted to approximately pH 7.0 by the addition of 750 l of 1 M Tris-HCl (pH 8.0) and frozen at ¹20⬚C until further use.
Enzyme assays The activity of key enzymes was determined according to the procedure described by Blasco et al. (1995). Highperformance liquid chromatography (HPLC) analyses were run according to the protocol described by Armengaud et al. (1998). 3-Chloro-cis,cis-muconate concentrations were calculated from measured cis-dienelactone concentrations because this compound is formed quantitatively on column from 3-chlorocis,cis-muconate under acidic HPLC conditions (Armengaud et al., 1998).
Lipid analysis Lipids were extracted using a modified Bligh–Dyer protocol, separated into polarity classes, and the fatty acids of the phospholipid fraction were converted to their methyl esters as described previously (Abraham et al., 1997). Capillary gas chromatographic analyses were performed on a Hewlett Packard 5890 series II gas chromatograph equipped with a capillary column HP Ultra 2 (5% diphenyl-, 95% dimethylpolysiloxane; 50 m; inner diameter 0.2 mm; film thickness 0.11 mm). The oven programme was 150⬚C for 2 min, 150⬚C to 289⬚C at 4⬚C min¹1 followed by an isothermal period of 11 min. Hexadecenoic methyl ester C16:1 comprised 9- and 11-hexadecenoic methyl esters (C 16:19 and C 16:17), which were not completely separated by GC-C-IRMS because of the requirement to handle very low amounts of substance. Therefore, both were determined as a single compound and referred to as C 16:1.
Fig. 3. Assignment of metabolic functions and interactions of members of the 4-chlorosalicylate metabolizing consortium. All metabolites shown for MT1 have been identified. The thick solid arrows indicate the experimentally determined release and fate of the corresponding metabolite in the community. The thick outline arrows indicate the experimentally determined release of small amounts of 3-chloromuconate whose fate has not been determined. The thin arrows indicate the possible release of further metabolites (see text). 䊚 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 167–174
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The fatty acid methyl esters were analysed for their isotopic ratios by means of a gas chromatograph coupled via a combustion/reduction interface to a Finnigan MAT 252 isotopic ratio mass spectrometer (GC-C-IRMS). Details of the analysis have been described elsewhere (Abraham et al., 1998).
Notation The standard notation for the expression of high-precision gas isotope ratio mass spectrometry results in the ␦ notation, defined as
␦ð‰Þ ¼ ððRFAME =RPDB Þ ¹ 1Þ¬ 103 where RFAME and RPDB are the 13C/12C isotope ratios corresponding, respectively, to the sample and to the international internal standard PeeDee belemnite, a South Carolinian carbonate rich in 13C. The more 13C a compound contains the higher ␦13C becomes.
Acknowledgements Christian Hesse is thanked for skilfully operating the IRMS, and Birgit Jung for technical assistance in the antibody experiments. The International Atomic Energy Agency, Vienna, Austria, is acknowledged for providing free reference materials for the calibration of the IRMS. This work was supported by grants from the German Federal Ministry for Science, Education and Research (project nos 0319433B and 0319433C). K.N.T. expresses gratitude to the Fonds der Chemischen Industrie for generous support.
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Blasco, R., Wittich, R.M., Mallavarapu, M., Timmis, K.N., and Pieper, D.H. (1995) From xenobiotic to antibiotic. Formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. J Biol Chem 49: 29229–29235. Blasco, R., Mallavarapu, M., Wittich, R.M., Timmis, K.N., and Pieper, D.H. (1997) Evidence that the formation of protoanemonin from metabolites of 4-chlorobiphenyl degradation negatively affects the survival of 4-chlorobi-phenyl-cometabolizing microorganisms. Appl Environ Microbiol 63: 427–434. Bru¨ckmann, M., Blasco, R., Timmis, K.N., and Pieper, D.H. (1998) Detoxification of protoanemonin by dienelactone hydrolase. J Bacteriol 180: 400–402. Chapman, P.A., Malo, A.T., Siddons, C.A., and Harkin, M. (1997) Use of commercial enzyme immunoassays and immunomagnetic separation systems for detecting Escherichia coli 0157 in bovine fecal samples. Appl Environ Microbiol 63: 2549–2553. Didry, N., Dubreuil, L., and Pinkas, M. (1991) Antibacterial activity of vapors of protoanemonin. Pharmazie 46: 546–547. Faude, U.C. (1995) Immunchemische Analyse mikrobieller Lebensgemeinschaften. PhD Thesis, University of Leipzig, Germany. Gibson, D.T., and Subramanian, V. (1984) Microbial degradation of aromatic hydrocarbons. In Microbial Degradation of Organic Compounds. Gibson, D.T. (ed.). New York: Dekker, pp. 181–252. Harms, H., Wilkes, H., Sinnwell, V., Wittich, R.M., Figge, K., Francke, W., et al. (1991) Transformation of 3chlorobenzofuran by Pseudomonas sp. HH 69. FEMS Microbiol Lett 65: 25–29. Mau, M., and Timmis, K.T. (1998) Use of subtractive hybridization to design habitat-based oligonucleotide probes for investigation of natural bacterial communities. Appl Environ Microbiol 64: 185–191. Morris, C.M., and Barnsley, E.A. (1982) The cometabolism of 1- and 2-chloronaphthalene by pseudomonads. Can J Microbiol 28: 73–79. Pelz, O., Hesse, C., Tesar, M., Coffin, R.B., and Abraham, W.R. (1997) Development of methods to measure carbon isotope ratios of bacterial biomarkers in the environment. Isotopes Environ Health Stud 33: 131–144. Seegal, B.C., and Holden, M. (1945) The antibiotic activity of extracts of Ranunculaceae. Science 101: 413–414. Tamura, G.S., Dailey, M.O., Gallatin, M.W., McGrath, M.S., Weissman, I.L., and Pillemer, E.A. (1984) Isolation of molecules by monoclonal antibodies and antisera: the solid phase immunoisolation technique. Anal Biochem 136: 458–464. White, D.C., Davis, W.M., Nickels, J.S., King, J.D., and Bobbie, R.B. (1979) Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40: 51–62.
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