1 Integrated Genomics, Inc., 2201 West Campbell Park Dr., Chicago, IL 60612, USA
2 Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83843, USA
3 The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1RQ, UK
4 Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612-7344, USA
Correspondence
Birgit M. Prüß
preuss{at}uic.edu
or
BirgitPruess{at}ndsu.nodak.edu
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ABSTRACT |
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Present address: Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58105, USA.
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INTRODUCTION |
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Recent work suggests multiple roles of FlhD/FlhC, some examples of which are the regulation of anaerobic respiration (Prüß et al., 2001) and the EntnerDoudoroff pathway in E. coli (Prüß et al., 2003
), synthesis of plasmid-encoded Yops (Bleves et al., 2002
) and phospholipase A (Schmiel et al., 2000
) in Y. enterocolitica and swarming in Serratia liquefaciens (Givskov et al., 1995
). FlhD alone is involved in regulating the cell division rate (Prüß & Matsumura, 1996
) through cadBA (Prüß et al., 1997
) and gltBD, gcvTHP and ompT expression (Prüß et al., 2003
) in E. coli.
To establish a global role of FlhD/FlhC in all enteric bacteria, bacteria other than E. coli will have to be studied. The FlhD and FlhC amino acid sequences of Y. enterocolitica and E. coli have an identity of 71·7 and 82·3 %, respectively. Y. enterocolitica FlhD/FlhC can complement an E. coli flhD mutant for motility (Young et al., 1999b). However, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli (Prüß et al., 2003). While the sequence similarity might indicate similarities in the regulatory function of FlhD/FlhC, the physiology of Y. enterocolitica differs significantly from E. coli. Y. enterocolitica is a human pathogen of the genus Yersinia, also containing the non-motile Yersinia pestis (causal agent of bubonic plague). In contrast to E. coli, Y. enterocolitica is able to grow at refrigerator temperatures. As a consequence, it is found in contaminated milk, milk products and pork. Y. enterocolitica shows phenotypic variation with respect to the growth temperature, including the synthesis of flagella and the production and secretion of virulence factors (Kapatral & Minnich, 1995
; Iriarte et al., 1995
; Kapatral et al., 1996
; Young et al., 1999a
, b
, 2000
; Young & Young, 2002
; Cornelis, 2002
).
In a first effort to investigate the global role of Y. enterocolitica FlhD/FlhC, we constructed a partial microarray and compared the expression patterns of wild-type cells with an flhD mutant at two different growth temperatures (25 and 37 °C). In a second set of experiments, the expression pattern of wild-type cells was compared at the two temperatures. A third temperature of 4 °C was used to study temperature profiles of selected genes and to address the psychrophilic character of Y. enterocolitica.
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METHODS |
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Bacterial strains and growth conditions.
The flhD flagellar gene of Y. enterocolitica strain 8081 was insertionally inactivated with the streptomycin cassette and cloned into a plasmid that contained the sacBR genes (Rimpilainen et al., 1992
). The vector was electroporated into Y. enterocolitica and integrated into the chromosome in a double-crossover event. The resulting strain (flhD : :
) was kanamycin- and sucrose-resistant. For reasons of laboratory safety, strains were devoid of the virulence plasmid. Overnight cultures of wild-type cells and the mutant strain were diluted 1 : 100 into 20 ml LuriaBertani broth (LB; 1 % tryptone, 0·5 % yeast extract, 1 % NaCl) in a 50 ml conical tube (Falcon). Cultures were grown at 4, 25 or 37 °C under constant shaking at 250 r.p.m. Under these growth conditions, both strains grew at a rate of 0·6 generations h1 during exponential growth when grown at 25 °C. At 37 °C, wild-type cells grew at a rate of 1·2 generations h1 and the mutants at 1·3 generations h1. At 4 °C, wild-type cells grew at a rate of 1·2 generations day1 and the mutants at 1·4 generations day1. At mid-exponential phase (OD600 of 0·5), cells were treated with 2 ml 5 % phenol in ethanol to prevent RNA degradation. Bacterial pellets were flash frozen in liquid nitrogen and stored at 70 °C.
For Phenotype MicroArrays, bacteria were grown overnight on R2A agar as recommended by the manufacturer (Biolog). For motility assays, bacteria were grown in tryptone broth (TB; 1 % tryptone and 1 % NaCl) to mid-exponential phase and 5 µl was spotted onto a tryptone swarm plate (0·3 % agar in TB). The plate was incubated in a humid environment at the indicated temperature. The diameter of the ring was measured over time.
RNA isolation, cDNA probe synthesis and hybridization conditions.
RNA was isolated by using the hot phenol/SDS method (Chuang et al., 1993). Final cleaning of the RNA was performed with an RNeasy mini column (Qiagen). Fluorescent cDNA probes were produced by amino-allyl reverse transcription and purified (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). Samples were dried and resuspended in 140 µl Sigma Arrayhyb hybridization solution (Sigma), supplemented with 2 µg yeast tRNA and subsequently combined with a complementary labelled cDNA. Hybridizations were performed in a GeneTAC HybStation (Genomic Solutions).
Data analysis, quality control and threshold determination.
Arrays were scanned on a GenePix 4000B Array Scanner (Axon Instruments) at 635 and 532 nm. Photomultiplier tube (PMT) voltages were adjusted to give the maximum signal from each channel without bleaching any of the features within the confocal image. For saturated spots, a second scan was performed at lower PMT voltage. Only spots that had a minimum expression level of 100 pixels in the high voltage scan were considered for analysis. Images were analysed using GenePix Pro 4.0 (Axon Instruments) as described previously (Prüß et al., 2003). The background was subtracted. Median intensities were used to determine expression ratios, which are higher than 1 for induced genes and below 1 for repressed genes. Due to the large number of genes on the array (2348 ORFs), it was possible to perform a normalization, using the total pixel values on each slide.
Each experiment was performed with RNA obtained from three independent bacterial cultures with a maximum of seven slides per experiment (the exact number of slides for each experiment are indicated in the table legends). Since the variation of the biological replicates (replicate cultures) was not any bigger than that of the technical replicates (replicate slides per culture), we determined the average of the mean and standard deviation for each predicted ORF across the entire population of data points (maximal 7 slides times 3 replicate spots equals 21 data points per ORF). Two statistical analyses were performed. Student's t-test compares the actual difference between two means in relation to the variation in the data. This is expressed as the standard deviation of the difference between the means (http://helios.bto.ed.ac.uk/bto/statistics/tress4a.html). Only predicted ORFs that had a t-test value less than 0·05 were considered for further analysis. The second statistical analysis was a determination of false positives, using Significance Analysis of Microarrays (SAM) (http://www-stat.stanford.edu/tibs/SAM; Tusher et al., 2001
). SAM assigns a score to each gene on the basis of a change in gene expression relative to the standard deviation of repeated measurements. A q-value was determined that is the lowest false discovery rate at which the gene is called significant. Only genes with a q-value less than 0·2 are included in Tables 1 and 2
.
To define a threshold level of significant difference, RNA from the wild-type strain grown at 25 °C was labelled with Cy3 and Cy5 and hybridized to a slide. Spot intensities of one sample were plotted against the spot intensities of the other sample (data not shown). Over 95 % of the data fell within the 1·8-fold range; more than 98 % were within twofold. An arbitrary significant expression ratio threshold was defined as twofold (expression ratio higher than 2 or lower than 0·5), which is consistent with published array work (Barbosa & Levy, 2000; Oshima et al., 2002
; Lehnen et al., 2002
; Lawhon et al., 2003
).
Only genes whose expression was greater than twofold or lower than 0·5-fold have been included in Tables 1 and 2. These genes are considered putative targets. Major conclusions were only drawn when at least one gene per operon was confirmed by quantitative PCR or differences were observed in Phenotype MicroArrays. This strategy is particularly important for genes whose expression ratio was close to the twofold cut-off.
Quantitative PCR.
Real-time PCR was performed with the same RNA samples used in the arrays. The PCR was performed with the SYBR Green kit from PE Biosystems. The reaction mixture contained 100 ng cDNA, 1x SYBR green buffer, 2·5 mM MgCl2, 0·25 mM each dNTP, 0·01 U AmpErase UNG, 1 U Taq Gold polymerase and 0·05 µM each primer. The primer pairings are available as supplementary data with the online version of this paper (at http://mic.sgmjournals.org). The reactions were performed at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min per cycle, for 50 cycles and were monitored in an iCycler iQ real-time PCR detection system (Bio-Rad). For each experiment, a standard curve was derived from plasmid pPM61 (Bartlett et al., 1988), expressing flhD. This was used to convert threshold crossings to log copy numbers. Expression ratios were obtained by dividing copy numbers of wild-type cells by those of the mutants. All PCR fragments yielded a single band on agarose gel.
Phenotype MicroArrays.
Phenotype MicroArrays (PMs) were obtained from Biolog. These are 96-well microtitre plates with a different cell culture medium dried to the bottom of each well (Bochner et al., 2001). We used PM3 (nitrogen sources) plates, to compare the phenotypes of wild-type cells with the flhD mutant. Standardized cell suspensions were produced using the 85 % turbidity standard and 100 µl was loaded onto each well. Growth was monitored with an EL311 Microplate Autoreader (BioTek Instruments) at 630 nm and analysed after 24 h. The OD630 of the wild-type culture was divided by the OD630 of the mutants. Growth ratios above 1 indicate nutrients that provided better growth conditions for wild-type cultures; growth ratios below 1 indicate nutrients that provided better growth conditions for the mutants. The growth of wild-type cells was compared in two independent experiments. Of 95 data points, 70 % were within one and 94 % within two standard deviations from the mean. A threshold level of a significant difference in growth was defined as growth ratio above 2 or below 0·5. Only nutrients that allowed one of the cultures to grow to an OD630 of at least 0·4 were considered significant.
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RESULTS |
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To investigate the regulation of some of the apparently unregulated flagella genes, real-time PCR was performed. The expression of cheY, flhA and fleC was regulated by FlhD/FlhC (twofold, 5·6-fold, and 12·7-fold, respectively). Altogether, we did not obtain any data contradicting the idea of FlhD/FlhC being the master regulator of flagella expression in Y. enterocolitica at 25 °C. FlhD/FlhC was not a regulator of flagella genes at the higher temperature of 37 °C.
The group of non-flagella genes that was maximally regulated by FlhD and FlhC at 25 °C were those involved in histidine degradation and carbamoylphosphate synthesis and conversion (Table 1). Among those were the genes encoding urocanate hydratase (hutU) and imidazolone kinase (hutI). Both enzymes are involved in the conversion of L-histidine to L-glutamate. The gene for histidine permease (hutP) was 10-fold induced. The pathway from L-histidine to L-glutamate requires two more enzymes, histidine ammonia lyase (hutH) and formylglutamate amidohydrolase (hutG). The hutH and hutG genes were not printed on the slides. However, real-time PCR analysis yielded an expression ratio of 2·5-fold for hutH and 5·6-fold for hutG. This indicates that the entire pathway is induced by FlhD and FlhC. Two more enzymes whose genes were induced by FlhD and FlhC were carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are involved in the synthesis of carbamoylphosphate and its conversion to carbamoylaspartate, which acts as a precursor for the synthesis of purines and pyrimidines. Within the metabolism of purines and pyrimidines, adenylate kinase (adk) converts AMP to ADP and dAMP to dADP.
To correlate gene regulation with phenotype expression, we compared growth patterns of wild-type cells and flhD : : mutants on Phenotype MicroArrays. Since most of the regulated genes were involved in nitrogen metabolism, PM3 (nitrogen sources) plates were used (Fig. 2
). The mutants grew better at 25 °C on 11 nitrogen sources, including six that are involved in the urea cycle. These are L-aspartate, L-arginine, L-citrulline, L-ornithine, agmatine and urea. In agreement with this, four urease genes were reduced by FlhD and FlhC (Table 1
). Even though the reduction in expression of these genes seems small, the expression of a phenotype suggests that the urea cycle is among the more important pathways that are regulated by FlhD and FlhC. In summary, the degradation pathway of L-histidine and the synthesis of carbamoylphosphate and carbamoylaspartate appear induced by FlhD and FlhC, whereas the urea cycle appears repressed (Fig. 3
). It is noteworthy that this regulation takes place only at 25 °C. At 37 °C, only hut genes and pyrBI are induced by FlhD and FlhC.
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Growth temperature affects the expression of FliA and FliZ but not FlhD/FlhC
To understand the differences between regulation by FlhD and FlhC at 25 and 37 °C (Table 1), we compared the expression pattern of wild-type cells at these two temperatures (Table 2
, column 4). Extending the temperature profile of gene regulation, a third temperature was added. The expression pattern of wild-type cells grown at 4 °C was compared to bacteria grown at 37 °C (Table 2
, column 5). The temperature profile for selected genes was determined across these three temperatures (Fig. 4
b and c).
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The temperature-regulated flagellar and chemotaxis genes are arranged in five operons: fliA (also containing fliZ), fleC, fliD (also containing fliS and fliT), motA (also containing motB, cheA and cheW) and tar (also containing tap, cheR, cheB, cheY and cheZ). The starting genes of these operons possess the putative binding consensus site [TAAA-(16)-GCCGATAA; Chilcott & Hughes, 2000] for FliA [fliA, position 69, tCaGATAA; fliD, position 63, TAAA-(15)-GCCGATAA; tar, position 108, gtAA-(16)-GCCGATAA; fleC, position 101, TtAA-(16)-GCCGATAc; motA, position 208, gtAA-(16)-GCCGATAt]. The fleC, fliD, motA and tar operons are considered class III operons in E. coli. With the exception of the fliA operon, regulation of class II operons could not be detected.
Real-time PCR was performed with genes of each operon to test the regulation of selected genes (Fig. 4a). Overall, microarray and real-time PCR data were consistent. Expression of flhD (class I) was not temperature-dependent. Expression of fliA and fliZ (class II) showed temperature regulation. Regulation of fleC, fliD, motA and cheY (class III) was confirmed.
Comparing the expression levels of flagella genes at 4 °C with those at 37 °C yielded inconsistent results (Table 2, column 5). The genes fliZ, motA and tar were the only ones that exhibited expression ratios above 2. Five other genes (fliD, cheY, cheZ, fliT and cheB) exhibited expression ratios between 1 and 2. These small expression ratios might demonstrate a lack of expression of flagella genes at all or indicate a beginning of an increase in expression, possibly followed by a larger increase later in growth. To distinguish between these two possibilities and to determine whether flagella are synthesized at any time during growth at 4 °C, we analysed the cells on tryptone swarm plates (Fig. 5
). Wild-type Y. enterocolitica cells were able to swarm at 4 °C with a maximal rate of 7·5 mm day1. At 25 °C, the maximal swarm rate was 48 mm day1. At 37 °C, the bacteria are fully non-motile. This indicates that Y. enterocolitica is indeed motile at 4 °C.
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Putative virulence factors that were differentially expressed are RYE00702 [araC-type transcriptional regulator ureBCFG (urease)], sodA and sodB (superoxide dismutase) and ompX (attachment and invasion).
Three regulators were expressed more than 10-fold higher at 4 °C than at 37 °C. These were a response regulator (RYE00701), the colanic acid regulator (rcsA) and RYE00702, encoding an araC-type transcriptional regulator. Also regulated at 4 °C were ampD (N-acetylmuramyl tripeptide amidase) and carAB (carbamoylphosphate synthetase). Repressed at 4 °C was ompX (outer-membrane protein).
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DISCUSSION |
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The enzyme that was most dramatically induced by FlhD and FlhC at 25 °C was urocanate hydratase (hutU). This enzyme was studied extensively as the key enzyme of the histidine utilization pathway (Fessenmaier et al., 1991; Kannan et al., 1998
). Other genes that are involved in this pathway that converts L-histidine to L-glutamate (Fig. 3
) are clustered in two operons. The first operon contains genes encoding histidine ammonia lyase (hutH) and histidine permease (hutP), in addition to urocanate hydratase. The second operon encodes the HutC repressor protein, imidazolone propionase (hutI) and formylglutamate amidohydrolase (hutG).
Once L-histidine is converted to L-glutamate, glutamate dehydrogenase can cleave it to 2-oxoglutarate and ammonia. Two enzymes, carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI), can fix ammonia through carbamoylphosphate into carbamoylaspartate. While carbamoylphosphate resembles the start of the urea cycle, carbamoylaspartate leads to the biosynthesis of purines and pyrimidines. In addition to the induction of carAB, pyrBI and the hut genes, we also observed a repression of four urease genes (ureBCFG). This repression was only about twofold and seemed too small to confirm with real-time PCR. However, Phenotype MicroArrays showed that flhD mutants grew two- to fivefold better on several intermediates of the urea cycle. The conclusion that the urea cycle might be regulated by FlhD/FlhC was based primarily upon this Phenotype MicroArray. Altogether, our data lead to the conclusion that FlhD and FlhC may balance the flow of nitrogen, using carbamoylphosphate as a checkpoint. Purine and pyrimidine biosynthesis are induced by FlhD and FlhC, while the urea cycle is repressed (Fig. 3).
The hypothesis that FlhD and FlhC may affect the levels of intracellular carbamoylphosphate (Fig. 3) offers another intriguing possibility. Response regulators (RR) of two-component systems are normally phosphorylated by their own cognate kinase (HK). However, small molecular mass phosphodonors such as carbamoylphosphate can act as substrates for the autophosphorylation of some RRs (Feng et al., 1992
). Given the impact that two-component systems have upon gene regulation in bacteria, the regulation by an alternative phosphodonor offers the cell an additional array of regulatory possibilities.
Since Y. enterocolitica FlhD and FlhC share significant homology with the E. coli orthologues, we expected an overlap between the FlhD/FlhC target gene lists. The genes that were regulated by FlhD/FlhC in both bacteria were carAB (carbamoylphosphate synthetase), tnaAB (tryptophanase and tryptophan permease; tnaLAB in E. coli), rbsD (ribose transporter; rbsA in E. coli), mglB (galactose transporter) and yfiD (pyruvate formate lyase). This equals 25 % of the genes regulated by Y. enterocolitica FlhD/FlhC. Besides this similarity, we observed differences. In E. coli, the majority of the FlhD/FlhC regulated genes was involved in aerobic and anaerobic respiration and the EntnerDoudoroff pathway (Prüß et al., 2003). Instead of regulating carbon metabolism, Y. enterocolitica FlhD and FlhC seems to be more involved in nitrogen metabolism by regulating amino acid degradation, purine/pyrimidine biosynthesis and the urea cycle.
Temperature affects the composition of the cell membrane and two-component systems
The expression patterns at three different temperatures were compared. To understand the differences between regulation by FlhD and FlhC at 25 and 37 °C (Table 1), we compared the expression pattern of wild-type cells at these two temperatures (Table 2
, column 4). An additional temperature of 4 °C (Table 2
, column 5) was chosen because Y. enterocolitica can cause fatal complications during blood transfusions when storing red blood cells at 4 °C and can act as a food pathogen in refrigerated food (Bradley et al., 1997
; Siblini et al., 2002
).
While incorporation of fatty acids of lower melting points has been discussed as a mechanism to restore membrane fluidity upon decreasing temperature (de Mendoza et al., 1993), membrane rigidification still takes place. This affects membrane-coupled processes, one of which is two-component systems. Among the genes that exhibited the highest increase in expression at 4 °C were two response regulators, RYE00701 and rcsA. Another response regulator, phoP, was 2·5-fold induced. This is in agreement with observations made in the cyanobacterium Synechocystis sp., where Hik33/Hik19 and Rer1 act as two-component systems for the perception and transduction of low temperature signals (Suzuki et al., 2000
) and the Gram-positive Bacillus subtilis, where the two-component system DesK/DesR fulfils a similar role (Aguilar et al., 2001
). The discovery of RcsA as a response regulator that is involved in temperature sensing is particularly striking because of its importance in biofilm formation and as a global regulator (Francez-Charlot et al., 2003
).
While the adaptation of the cytoplasmic membrane is well investigated and understood, little is known about the impact on the outer membrane of Gram-negatives. It has been observed, however, that OmpF and OmpC are regulated in an inverse manner. In agreement with this, ompF was expressed higher at 25 and 4 °C than at 37 °C. We could not detect an effect of growth temperature on ompC expression. Instead, we identified two more outer-membrane proteins, whose genes were regulated by temperature (ompW and ompX).
Temperature regulation of class III flagella genes might be mediated by FliA
A number of interesting observations were made analysing temperature-dependent expression of flagella genes (25 vs 37 °C). The hierarchy of flagella expression is believed to be similar to E. coli (Aldridge & Hughes, 2002). FlhD/FlhC was described as the flagella master regulator at 26 °C (Young et al., 1999b
), which is consistent with our microarray data (Table 1
). In contrast, the environmental control of the master regulator may differ. In E. coli, all the known regulators that affect the expression of flagella genes connect through FlhD/FlhC (Prüß & Wolfe, 1994
; Shin & Park, 1995
; Wei et al., 2001
; Lehnen et al., 2002
; Francez-Charlot et al., 2003
). This study demonstrates a constitutive expression of the Y. enterocolitica flagella master operon with respect to growth temperature. To our knowledge, this has not been shown yet. It suggests that temperature regulation of flagella genes in this bacterium might not be mediated by changes in the expression level of flhD.
It has been previously shown that the class II and class III flagella genes of Y. enterocolitica do not all respond to the same environmental stimuli. For example, the flagellin genes fleA, fleB and fleC (Kapatral & Minnich, 1995), the genes encoding the flagella-specific sigma factor fliA and the anti-sigma factor flgM (Kapatral et al., 1996
) were regulated by temperature. In contrast, the expression of flhB, flhA and flhE was demonstrated to be temperature independent (Fauconnier et al., 1997
). This study confirms and extends these observations. The expression of four class III operons (fleC, fliD, motA and cheY) was affected by temperature. All of these contained the putative binding site for FliA. The data are consistent with the hypothesis that the block in motility at 37 °C might be at the level of fliA and not flhD expression.
Temperature regulation of motility is an important factor in bacterial pathogenicity. For example, flagellin is a potent cytokine inducer (McDermott et al., 2000) and its repression may contribute to the observed non-inflammatory response characteristic of Yersinia infections (Cornelis, 2000
). Consequently, permanent loss of FlhD/FlhC is displayed by a wide range of Gram-negative pathogens, including Y. pestis (Parkhill et al., 2001
; Deng et al., 2002
). However, FlhD/FlhC has numerous other functions and its repression could come at a significant cost for the bacteria. Performing temperature regulation of flagellin synthesis at the level of FliA may confer advantages, such as the maintenance of numerous non-flagella functions of FlhD/FlhC.
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ACKNOWLEDGEMENTS |
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Received 3 October 2003;
revised 6 January 2004;
accepted 25 March 2004.
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