INTRODUCTION

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Proteomics is a newly emerging research field which allows the analysis of when and under what conditions gene-encoded events (e.g., protein translation) occur (3, 11, 24). Proteome analysis by two-dimensional gel electrophoresis (2-DE) has been proposed elsewhere as a powerful tool for making genomics functional (3, 12, 21). One of the cornerstones for making proteomics a powerful tool is the development of mass spectrometry supported by the matrix-assisted safe ionization of peptide fragments and delayed extraction for the purpose of enhancing resolution power (22, 23). These extended capabilities of mass spectrometry, along with the ever-increasing amount of protein sequence data in various databases, are making protein identification and the characterization process a feasible task.

Poly(3-hydroxybutyric acid) (PHB) is an intracellular carbon and energy storage material synthesized by numerous microorganisms, usually when growth is impaired by the depletion of a specific nutrient in the presence of excess carbon source (13, 14, 33). PHB has been drawing much attention because of its complete biodegradability and the possibility of producing it from renewable resources (13, 14). In Ralstonia eutropha and Alcaligenes latus, PHB is synthesized from acetyl coenzyme A (CoA) in three sequential reaction steps catalyzed by -ketothiolase, acetoacetyl-CoA reductase, and PHB synthase (13, 16, 30). The second reaction catalyzed by the reductase requires NADPH as a cofactor. A metabolically engineered Escherichia coli strain constitutively expressing the heterologous PHB biosynthesis genes has been suggested elsewhere to be a good candidate for PHB production due to fast growth, a large amount of PHB accumulation, and the availability of well-established high-cell-density culture techniques (8, 13, 18). Even though recombinant E. coli has been successfully employed for the high-level production of PHB (37), whether the overall cellular physiology is altered due to the expression of heterologous PHB biosynthesis genes and the accumulation of PHB granules in the cytoplasm remains unclear.

In this study, we analyzed and compared the proteomes of a metabolically engineered E. coli strain under PHB-producing and non-PHB-producing conditions. Proteome expression patterns of recombinant E. coli were resolved on 2D gels, and the variations in the relative expression levels of particular proteins were examined using a software-aided protein quantification tool.

	MATERIALS AND METHODS

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Bacterial strain, plasmid, and growth condition. The E. coli strain used in this study was XL1-Blue (supE44 hsdR17 recA1 gyrA96 thi relA1 lac F' [proAB⁺ lacl^q lacZM15 Tn10(Tet^r)]). Plasmid pJC4, which contains the A. latus PHB biosynthesis genes, has been described previously (8). The PHB biosynthesis genes are constitutively expressed in E. coli (8). However, these enzymes cannot be detected on the 2D gel due to a low expression level (31). As a control plasmid, pJC4phb was constructed by deleting the PHB operon from pJC4. E. coli XL1-Blue, recombinant E. coli XL1-Blue(pJC4), and recombinant E. coli XL1-Blue(pJC4phb) were grown to early stationary phase in 250-ml flasks containing 100 ml of Luria-Bertani (LB) medium or LB medium plus 20 g of glucose per liter in a shaking incubator at 200 rpm at 37°C. Ampicillin was added at a concentration of 50 mg/liter when cultivating recombinant E. coli XL1-Blue(pJC4) and recombinant E. coli XL1-Blue(pJC4phb).

Analytical procedures. Cell growth was monitored by measuring the absorbance at 600 nm (optical density at 600 nm; DU Series 600 spectrophotometer; Beckman, Fullerton, Calif.). The PHB concentration was determined by measuring the concentration of 3-hydroxybutyric acid methyl ester, which was prepared by methanolysis of PHB, with a gas chromatograph (Donam Co., Seoul, Korea) equipped with a fused silica capillary column (Supelco SPB-5; 30 m by 0.32 mm in inside diameter; 0.25-µm film; Bellefonte, Pa.) using benzoic acid as an internal standard (6). The cell concentration, defined as dry cell weight per liter of culture broth, was determined as previously described (37). The PHB content (wt%) was defined as the percent ratio of PHB concentration to cell concentration.

2-DE. The 2-DE was carried out using a Protean II xi 2-D cell (Bio-Rad Laboratories) following the procedures described previously (10, 12) with slight modifications as follows. Carrier ampholyte solutions having a 4:1 ratio (vol/vol) of Bio-lyte pH 5 to 7 to Bio-lyte pH 3 to 10 (Bio-Rad) were used for the formation of a pH gradient in an isoelectric focusing (IEF) tube gel. Culture broth was centrifuged for 5 min at 3,500 × g and 4°C. The pellet was washed four times with TE solution (10 mM Tris-HCl, 1 mM EDTA; pH 8.0) and was resuspended in double-distilled water followed by four cycles of sonication (each for 10 s at 10% of maximum output; high-intensity ultrasonic liquid processors; Sonics & Material, Inc., Newtown, Conn.). Soluble protein was obtained by the centrifugation of cell extract at 10,000 × g and 4°C for 20 min. After the protein quantification by the Bradford assay using bovine serum albumin as a standard (5), protein samples (300 µg) were dried down by vacuum centrifugation, suspended in IEF denaturation buffer [9 M urea, 0.5% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 10 mM dithiothreitol, 0.2% (wt/vol) Bio-lyte pH 3 to 10, 0.001% (wt/vol) bromophenol blue; final volume, 200 µl], and were carefully loaded into the IEF tube gel with a syringe. Then, the loaded tube gels were placed on sodium dodecyl sulfate-12% polyacrylamide gels prepared by a standard protocol (12). Coomassie brilliant blue R-250 (Bio-Rad) was used for protein staining (10). After overnight destaining in a solution composed of 40% (vol/vol) methanol and 10% (vol/vol) acetic acid, gels were scanned using a GS710 calibrated imaging densitometer (Bio-Rad). Melanie II software (Bio-Rad) was used to automate the process of finding protein spots within the image and to quantify the density of the spots on a volume basis (i.e., values were calculated from the integration of spot optical intensity over the spot area). To check the reproducibility and to estimate standard deviations, protein samples taken from duplicate cultures were analyzed in duplicate 2-D gels.

Peptide mass fingerprinting. Samples for the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis were prepared as described previously (28) with some modifications described below. Trypsin digestion was carried out overnight at 37°C in a stationary incubator. A relatively high concentration of trypsin (25 ng/µl; sequencing grade; Boehringer Mannheim Co., Mannheim, Germany) was used to enhance trypsin autolysis. Two standard peaks of 805.417 (m/z) and 2,163.056 (m/z) were generated from this trypsin autolytic reaction. Other peptide fragment peaks, which are usually placed between these two standard peaks, were calibrated using the values of these two peaks. In-gel-digested peptide fragments were extracted from gel pieces by the addition of 100 µl of 60% (vol/vol) acetonitrile and 0.1% (vol/vol) trifluoroacetic acid (TFA) solution, followed by vortexing for 1 h. After the transfer of supernatant solution into a new Eppendorf tube, the acetonitrile-TFA solution was added for the final extraction of remaining peptide fragments. After these two supernatant solutions were combined, solute materials including peptide fragments were dried down by vacuum centrifugation. To eliminate impurities, such as salt molecules, gel particles, and traces of Coomassie brilliant blue, the samples were passed through the ZipTip column (Millipore Co., Bedford, Mass.) in which C₁₈ resin is fixed at the end of the tip. -Cyano-4-hydroxycinnamic acid, saturated in a solution composed of 50% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA, was used as a matrix for MALDI-TOF. This matrix was incorporated with contaminant-free peptide fragments, placed on the sample plate, and crystallized with the peptide sample by air drying. The MALDI-TOF mass spectrometry system used was the Voyager Biospectrometry system (PerSeptive Biosystems, Inc., Framingham, Mass.). Laser intensity for the ionization of samples was optimized between 2,400 and 2,600. The accelerating voltage for the flight of ionized particles was 21,000 V, and the delayed extraction time was 150 ns.

	RESULTS

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Cell growth and PHB accumulation. Flask cultures of recombinant E. coli XL1-Blue(pJC4) were grown in LB medium or LB medium containing 20 g of glucose per liter. Recombinant E. coli XL1-Blue(pJC4) could efficiently produce PHB in the latter, while it did not in the former (8). Flask cultures of E. coli XL1-Blue without the plasmid and of recombinant E. coli XL1-Blue(pJC4phb) were also made as controls. Table 1 shows the cell and soluble protein concentrations at the time of harvesting (early stationary phase) for proteome analysis. The PHB concentration and PHB content obtained for XL1-Blue(pJC4) in LB medium plus 20 g of glucose per liter at the time of proteome analysis were 0.89 g/liter and 68%, respectively. For both wild-type and recombinant strains, the acetic acid concentrations at the time of harvesting were 0 and 2 g/liter when grown in LB medium and LB medium plus 20 g of glucose per liter, respectively.

Proteome analysis. Proteome expression profiles of XL1-Blue and XL1-Blue(pJC4) grown in two media (LB medium and LB medium plus 20 g of glucose per liter) are shown in Fig. 1. To compare the relative expression levels of cellular proteins, the similar amounts of proteins were loaded on 2D gels. Cellular proteins possessing pIs between 4.7 and 6.2 could be nicely separated in our 2D gels. The overall profiles of synthesized proteins within this range were quite reproducible and distinctive enough to be compared and matched even by naked eyes.

View larger version (102K): [in this window] [in a new window]   FIG. 1.   Proteome expression profiles of E. coli XL1-Blue grown in LB medium (A), E. coli XL1-Blue grown in LB medium plus 20 g of glucose per liter (B), recombinant E. coli XL1-Blue harboring pJC4 grown in LB medium (C), and recombinant E. coli XL1-Blue harboring pJC4 grown in LB medium plus 20 g of glucose per liter (D). The horizontal axes represent the isoelectric points, and the vertical axes represent the molecular masses in kilodaltons.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Quantification of identified protein spots showing altered expression level from Fig. 1. Spot intensities were measured and normalized as described in Materials and Methods. Error bars represent the standard deviations. Vol% is the percentage of relative values calculated from the integration of spot intensity over the spot area. For the symbol key, top to bottom corresponds to left to right for each group of bars.

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	DISCUSSION

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Recombinant E. coli harboring a plasmid containing the heterologous PHB biosynthesis genes was found to be able to accumulate a large amount of PHB (8, 13, 18, 37). It was expected that this metabolically engineered E. coli strain would undergo physiological changes upon the accumulation of PHB, which is not a normal metabolite of E. coli. We attempted to examine these physiological changes by analyzing the proteomes of recombinant E. coli XL1-Blue under two different culture conditions: PHB-producing and non-PHB-producing conditions. The proteomes of XL1-Blue without the plasmid and XL1-Blue harboring backbone plasmid were also analyzed for comparison.

Glucose effect. EF-Tu is one of the most abundant cytosolic proteins and plays a crucial role in protein biosynthesis. It was reported previously that EF-Tu interacts with various cellular macromolecules such as tRNAs charged with amino acids; another type of protein chain elongation factor, EF-Ts; and ribosomes to make the translational process proceed properly (35). According to Fig. 1 and 2, it can be seen that the expression level of EF-Tu decreased significantly in cells grown in the presence of glucose. Since cellular protein synthesis is reduced, more catabolic intermediates seem to be available for PHB biosynthesis under this condition. This was supported by the finding that a large amount of PHB was accumulated only when glucose was present in the medium (17, 18). The synthesis of Dps was twofold greater in the presence of glucose. Dps has been shown previously to be expressed when cells produce acetic acid (27, 36). This is in agreement with our finding that 2 g of acetic acid per liter was produced only in the presence of glucose.

PHB accumulation effect. In general, heat shock proteins are synthesized in order to protect cells from external stresses such as sudden increase of temperature, UV irradiation, virus infection, organic solvents, and others. Expression of heterologous genes can also trigger heat shock response (9). Based on our experimental observation of increased expression of three heat shock proteins (GroEL, GroES, and DnaK) in PHB-producing cells, PHB accumulation in recombinant E. coli can be considered as a stress on the cells inducing heat shock response. Furthermore, the takeover of the cytosolic space by PHB granules would disturb the normal intracellular architecture such as chromosome attachment and consequently would result in heat shock response. Bacteria naturally accumulating PHB synthesize phasin protein, which covers the surface of PHB granules. E. coli does not naturally produce PHB and therefore does not have the phasin gene (39). Therefore, the hydrophobic PHB granules are in direct contact with intracellular biomolecules including DNA, RNA, and proteins. Obviously, this will become a major stress on the cells for several possible reasons including denaturation of proteins on the surface of PHB granules. This unfavorable condition generated by PHB accumulation along with the reduced synthesis of EF-Tu seems to have resulted in the further reduction of protein synthesis.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Central metabolic pathways of recombinant E. coli producing PHB. Several competing metabolic pathways leading to the synthesis of PHB, acetate, citrate, and fatty acids from acetyl-CoA are shown. Four identified enzymes, Fba, TpiA, GpmA, and Eda, and the reaction steps that they catalyze are shown in boldface. The reaction pathways leading to the synthesis of PHB along with the enzymes involved are also shown in boldface.

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