1 UFZ Centre for Environmental Research Leipzig-Halle, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany
2 University of Aberdeen, Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
Correspondence
Dirk Benndorf
dirk.benndorf{at}ufz.de
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ABSTRACT |
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The protein sequence data reported in this article will appear in the SWISS-PROT and TrEMBL knowledgebase under accession numbers Q8KN28, Q9RNZ9, Q93T12, Q9R5K5, P83709, P83707, P83710, P83712, P83708 and P83711.
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INTRODUCTION |
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However, the presence of genetic information is not sufficient to guarantee biodegradation of chlorophenoxy acids at strongly contaminated sites, since micro-organisms may be poisoned by growth substrate or metabolites, if the concentrations are high and the substances are potentially toxic. First, chlorophenoxy acids are hydrophobic compounds and weak organic acids; therefore, they are potentially toxic agents which may disturb the energy conservation system of bacteria located in the cytoplasmic membrane (Loffhagen et al., 1997). Second, the lipophilicity and the reactivity of compounds may cause damage to several biomolecules as described in Acinetobacter calcoaceticus, which was concluded from the enhanced synthesis of heat-shock proteins and oxidative-stress proteins (Benndorf et al., 1999
, 2001
). However, the presence of the chlorophenoxy herbicide 2,4-dichlorophenoxypropionic acid (2,4-DCPP) and its metabolites 2,4-dichlorophenol (2,4-DCP) and 3,5-dichlorocatechol during growth of Delftia acidovorans MC1 (Benndorf & Babel, 2002
) on pyruvate caused no significant induction of the respective proteins, whereas the induction of catabolic enzymes indicates that productive detoxification is also a component of the response to chemostress in a bacterium which is able to metabolize these compounds (Benndorf & Babel, 2002
).
The goal of the present research is to elucidate and understand the long-term adaptation of D. acidovorans strain MC1 during continuous growth on low and high concentrations of chlorophenoxy herbicides simulating both low bioavailability and excess concentration of substrates at contaminated sites. Proteome analysis has been proven to deliver an overall picture of gene expression that represents the metabolic state of an organism (Peng & Shimizu, 2003), as well as indicating the presence of adaptive responses (Vasseur et al., 1999
). It was used in this study as this technique also allows recognition of post-translational modifications which may also play a role in response(s) to chemostress.
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METHODS |
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For growth on excess concentrations of 2,4-DCPP, the pH-auxostat principle (Krayl et al., 2003) was used. Briefly, the reservoir medium containing the substrate was used to adjust the pH of the culture. Several concentrations between 4·2 and 29·5 mM 2,4-DCPP and different volumes of 2 M KOH were added to the medium, whereas the buffer capacity of the medium had to be lower than the amount of acid which would be released if all 2,4-DCPP were consumed. For batch experiments, D. acidovorans MC1 was pre-cultivated on minimal medium pH 7 containing 3 g sodium pyruvate l1 in batches, overnight. Then, 5 ml inoculum was added to 20 ml medium (pH ranging between 6 and 8·5) containing 3 g sodium pyruvate l1 and several concentrations of 2,4-DCPP.
Growth was measured spectrophotometrically by monitoring optical density at 700 nm.
Determination of 2,4-DCPP, its metabolites and measurement of enzyme activities.
Concentrations of 2,4-DCPP and 2,4-DCP were determined by HPLC analysis (Oh & Tuovinen, 1990). The activities of chlorocatechol 1,2-dioxygenase (Müller et al., 2001
) and dichlorprop/2-oxoglutarate dioxygenases were measured as described previously (Westendorf et al., 2003
).
Sample preparation, 2D electrophoresis and electroblotting.
The bacteria were harvested and the cells were lysed as described previously (Benndorf & Babel, 2002). The protein content was determined as described previously (Holtzhauer & Hahn, 1988
). For 2D electrophoresis, 50 µg protein for analytical gels and 2000 µg for micropreparative gels were precipitated with ice-cold acetone, resolubilized, loaded on 18 cm long Immobiline DryStrip pH 310 NL and 2D electrophoresis was carried out as described previously (Benndorf & Babel, 2002
). Analytical gels were silver-stained as described by Blum et al. (1987)
and dried in a stream of unheated air from a GelAir Dryer (Bio-Rad). With respect to reproducibility, we carried out two independent experiments with at least two gels per sample. The best gels of each replicate were selected for image analysis. For mass spectrometry (MS) and internal protein sequencing, micropreparative gels were stained with Coomassie blue, whereas for amino terminal sequencing, the gels were first electroblotted overnight on PVDF (Bio-Rad) membranes using the CAPS buffer system (Jin & Cerletti, 1992
) and stained with Coomassie blue afterwards (Benndorf & Babel, 2002
).
Identification of proteins by amino terminal and internal sequencing and MS.
For peptide-mass mapping, proteins of interest were excised from micropreparative 2D gels digested by trypsin in-gel and analysed using a Voyager-DE STR MALDI-TOF mass spectrometer at the Aberdeen Proteome Facility (Cash et al., 1999). Furthermore, proteins were identified by amino terminal and internal amino acid sequencing using a model 491cLC protein sequencer (Applied Biosystems). Before internal sequencing, the protein spots were excised from micropreparative 2D gels and digested in-gel as described above except that Lysyl Endopeptidase (LysC) from Achromobacter lyticus (Wako Chemicals; 2 µg in 200 µl 25 mM Tris/HCl pH 8·0) was used instead of trypsin. The peptides were separated by HPLC using a self-packed column (column length 150 mm, i.d. 0·5 mm, Self Pack POROS 10 R2 Reversed Phase Packing, PerSeptive Biosystems). A gradient with a flow rate of 50 µl min1 and with increasing acetonitrile concentrations was used (buffer A 0·1 % TFA in water; buffer B 0·085 % TFA in 70 % acetonitrile, increase from 0 % B to 60 % B within 90 min). The peptides were detected by UV absorption at 214 nm and fractions containing peptides were collected manually. The fractions were dried down to 10 µl, mixed with 100 µl of 0·1 % TFA in water and applied to Prosorb cartridges (Applied Biosystems) according to the manufacturer's instructions.
Comparison of 2D protein patterns.
Silver-stained dried gels were scanned using a UMAX Power Look 2000 Scanner with an eight bit dynamic range and 200 d.p.i. resolution. Gel images were analysed by PHORETIX 2D 5.01 software (NonLinear Dynamics). For comparing protein patterns, we used only spots that were present in both gels of replicated experiments. Protein spots with a twofold, or greater, normalized volume (density) than the corresponding spots in control gels were considered to be amplified, and spots with half, or less, normalized volume than the corresponding spots in control gels were considered to be diminished. Spots which were observed following imposition of stress conditions, but not in the control protein pattern, were considered to be newly synthesized proteins. Molecular mass and pI were calibrated using internal standards defined by calibration with the 2D SDS-PAGE Standards kit (Bio-Rad).
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RESULTS AND DISCUSSION |
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Afterwards, the protein pattern after chemostatic growth on 2,4-DCPP representing low residual concentration was taken as a control pattern for comparison with growth at high residual concentration of 2,4-DCPP (pH-auxostat regime). Seventy-four changed protein spots (34 amplified, 22 newly synthesized, 18 diminished, see Fig. 2) indicate that D. acidovorans MC1 is able to adapt at the protein level to excess 2,4-DCPP. To measure the dynamics of protein pattern in response to 2,4-DCPP, protein samples were also taken from the time-course experiment with instant addition of 15 mM 2,4-DCPP and 0·5 mM 2,4-DCP. The corresponding 2D gels (whole gels not shown) showed that induction profiles of the identified proteins were very different (Figs 3
and 4). The further induction of catabolic enzymes (chlorocatechol 1,2-dioxygenase TfdCII, chloromuconate cycloisomerase TfdD) can probably increase the rate of biodegradation and reduce the concentrations of metabolites. Furthermore, the activities or specificities of some enzymes, particularly of TfdD, are additionally regulated by post-translational modifications because the occurrence of isoforms 3 and 4 was favoured in the presence of excess 2,4-DCPP (Figs 4 and 5
). However, an inactivation of enzymes resulting from chemical reactions with reactive metabolites, for example 3,5-dichlorocatechol or others, cannot be excluded, since the culture was sometimes brownish coloured. However, 3,5-dichlorocatechol was never detected by HPLC. Moreover, the SdpA synthesis was repressed in the presence of high concentration of 2,4-DCPP. On the one hand, its decrease after one day reveals a potential bottleneck because this enzyme catalyses the first step of biodegradation of the S-enantiomer of 2,4-DCPP. On the other hand, the decrease of this enzyme, which was confirmed by the measurement of specific activity in cell-free extracts (Table 4
), may indicate that the enzymic release of the more toxic 2,4-DCP from 2,4-DCPP was reduced. A detailed analysis of the induction pattern and of the transcription is necessary to clarify if the decrease happens due to less stability of the enzyme or due to decreased transcription of the sdpA gene.
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As early as 1 h after the addition of high concentrations of 2,4-DCPP TufA was modified by post-translational modification. Its low molecular mass isoform began to disappear whereas its high molecular mass isoform appeared (Figs 3 and 5). TufA is an essential part of the protein synthesis apparatus, promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes and seems to be implicated in protein folding and protection from stress, because it also shows chaperone activity in vitro (Caldas et al., 1998
). More than one isoform of TufA was found in 2D databases of Escherichia coli (SWISS-2DPAGE, http://us.expasy.org/ch2d/) and Mycobacterium tuberculosis (European Bacteria Proteome Project, http://www.mpiib-berlin.mpg.de/2D-PAGE/EBP-PAGE/index.html). A proposed mechanisms for modification is proteolytic cleavage of TufA in E. coli as response to phage T4 infection (Georgiou et al., 1998
) and in Salmonella typhimurium as response to starvation of phosphate (Adams et al., 1999
). Furthermore, regulation of TufA in response to nutrient deprivation by methylation (Young & Bernlohr, 1991
) or phosphorylation of TufA (Lippmann et al., 1993
) was described in E. coli. Probably, TufA is proteolytically cleaved at its carboxy terminal end, because both isoforms are blocked at the amino terminal end as also described in E. coli (Jones et al., 1980
) and a limited proteolysis seems to be more probable in bacteria than a glycosylation with an estimated mass of more than 2000 Da. However, our data are insufficient to identify the site and kind of modification of TufA in D. acidovorans MC1 as well as its effect on metabolism. As reported by other authors, proteome analysis often delivers much data (Blom et al., 1992
) which are difficult to interpret, for example the bulk of unidentified spots (more than 60). In addition, the identification of such stress protein sometimes brings up more questions because proteins such as TufA, AspG, OdhB and YceI are involved in complex metabolic and regulatory networks.
Conclusion
Finally, proteome analysis allowed us to evaluate the response of D. acidovorans MC1 to 2,4-DCPP on a global level. One strategy of adaptation during growth on high residual concentrations of 2,4-DCPP could be the repression (SdpA) as well as the induction (TfdCII, TfdD) of catabolic enzymes probably providing resistance by lowering the concentrations of toxic intermediates. Classical stress proteins play a minor role. Furthermore, the modification of essential proteins such as TufA and the induction of the hypothetical periplasmic protein YceI indicate that further important mechanisms of resistance may exist. Their investigation is indispensable for a comprehensive understanding of the response of bacteria to chlorophenoxy herbicides.
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ACKNOWLEDGEMENTS |
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Received 18 September 2003;
revised 24 November 2003;
accepted 28 November 2003.
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