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Applied and Environmental Microbiology, May 2003, p. 2521-2532, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2521-2532.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of L-Valine

C. Lange, D. Rittmann, V. F. Wendisch,* M. Bott, and H. Sahm

Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany

Received 24 October 2002/ Accepted 31 January 2003


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ABSTRACT
 
Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 {Delta}ilvA {Delta}panBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.


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INTRODUCTION
 
Corynebacterium glutamicum was isolated as an L-glutamate-excreting bacterium (1) and is used today for the large-scale biotechnological production of two amino acids, i.e., L-glutamate (800,000 tons per year), a flavor enhancer, and L-lysine (400,000 tons per year), which is mainly used as a feed additive (17). In contrast to glutamate, lysine can be produced only by an appropriate mutant strain of C. glutamicum, e.g., MH20-22B (50), which accumulates 230 mM L-lysine in the culture medium. The use of C. glutamicum for lysine production is particularly advantageous, since it is a "GRAS" (generally regarded as safe) organism, and therefore, downstream processing of lysine does not require separation from the biomass.

For a number of reasons, C. glutamicum is very well suited for the production of additional amino acids. (i) The regulation of biosynthetic pathways is often less complex in C. glutamicum than in many other bacteria. For example, C. glutamicum possesses only one acetohydroxyacid synthase (AHAS) (28), while Escherichia coli contains three differently regulated isoenzymes (3, 56). (ii) Central carbon metabolism, anaplerotic reactions, and several amino acid biosynthesis pathways, as well as many transport processes, have been studied in great detail in C. glutamicum (45). (iii) The genome sequence is known, and effective tools for genetic manipulation are available.

By metabolic engineering, C. glutamicum strains that produce L-valine have been created (41). This branched-chain amino acid is essential for vertebrates, and its production is of commercial interest because of its use as a feed additive, for infusion solutions, and as a precursor for the chemical synthesis of herbicides (17, 34). Currently, ~500 tons of L-valine is produced per year by fermentation or extraction from acidic hydrolysates of proteins (17). As shown in Fig. 1, valine is synthesized from two molecules of pyruvate in a pathway involving four reactions which are catalyzed by AHAS (the ilvBN gene product), isomeroreductase (the ilvC gene product), dihydroxyacid dehydratase (the ilvD gene product), and transaminase B (the ilvE gene product). As in other organisms, the same enzymes also catalyze the synthesis of L-isoleucine from pyruvate and 2-ketobutyrate. The latter is formed from L-threonine by threonine dehydratase (the ilvA gene product). AHAS is the key enzyme of branched-chain amino acid synthesis. This enzyme is feedback inhibited by valine, leucine, and isoleucine, but even in the presence of all three amino acids, the activity is inhibited maximally to ~50% (16, 19). In E. coli, the effect of valine on its three AHAS isoenzymes has been described (54). Valine causes feedback inhibition of AHAS I, encoded by ilvBN, and AHAS III, encoded by ilvIH (13), and their small regulatory subunits, IlvN and IlvH, were shown to be necessary for valine sensitivity (6, 7, 20). In contrast, AHAS II, encoded by ilvGM, is resistant to valine (4, 13, 24, 54). In the presence of valine, the lack of ilvGM expression in E. coli K-12 causes a growth defect due to 2-ketobutyrate toxicity and leucine and isoleucine limitation (13, 24, 31, 54, 59). Valine-resistant mutants of E. coli K-12 showed restored ilvGM expression (31). In E. coli, valine regulates the expression of ilvBN, as well as ilvGMEDA, by an attenuation mechanism (14, 21, 25, 32, 54), whereas ilvIH expression is controlled by the leucine-responsive protein Lrp (43, 54, 55). In C. glutamicum, the synthesis of AHAS is regulated as well. Expression of ilvBN is altered about twofold in response to the branched-chain amino acid concentration via an attenuation mechanism (38). In contrast to a direct amino acid deficiency, a valine, leucine, and pantothenate shortage due to ketobutyrate addition led to ~10-fold-increased AHAS activity, probably caused by an additional as-yet-unknown control (16, 38).



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  		FIG. 1. Biosynthetic pathways of L-valine, L-isoleucine, L-leucine, and D-pantothenate in C. glutamicum. The solid arrows indicate reactions catalyzed by the indicated enzymes, and the dotted arrows indicate multistep pathways. Relevant gene names are given in parentheses.

The C. glutamicum valine production strain 13032 {Delta}ilvA {Delta}panBC(pJC1ilvBNCD) (called VAL1 hereafter), derived from the wild type by metabolic engineering, excretes up to 90 mM L-valine (41). This was made possible by a number of defined genetic alterations. (i) The valine biosynthesis genes ilvB, ilvN, ilvC, and ilvD were overexpressed from a plasmid. (ii) The chromosomal ilvA gene was deleted in order to avoid the formation of isoleucine as a major by-product. In addition, cultivation of the strain under isoleucine limitation could increase ilvBN expression by the attenuation mechanism (38). (iii) The panBC genes encoding two steps in the D-pantothenate synthesis pathway were deleted. Pantothenate is a constituent of coenzyme A (CoA) (27) and thus is required for the oxidative decarboxylation of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex. Cultivation under pantothenate limitation will limit this reaction and thereby increase the availability of the valine precursor pyruvate.

Within a project aiming to improve valine production by further metabolic engineering of the VAL1 strain, the question arose as to what effects the presence of valine itself would have on growth and valine production. This question was triggered by the observation that addition of 30 mM L-valine or L-isoleucine to the culture medium significantly reduced the growth rate of the lysine producer C. glutamicum MH20-22B (18). It was speculated that the hydrophobicity of these branched-chain amino acids was somehow responsible for the deleterious effect, because the hydrophilic amino acids L-alanine and L-lysine caused no inhibition. In addition, mischarging of tRNA has been discussed as an alternative explanation (18). The inhibitory effect could also be responsible for the generally lower levels of production by C. glutamicum of nonpolar amino acids like valine compared to polar amino acids like lysine (34). Since the mechanism of growth inhibition of C. glutamicum MH20-22B by valine and isoleucine remained unexplained, we decided to study the influence of valine on wild-type C. glutamicum ATCC 13032 and on the valine production strain VAL1 by combining growth studies with transcriptome and proteome analyses.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, media, and growth conditions.
The bacterial strains and plasmids used in this work are listed in Table 1. For the first preculture, 5 ml of CGIII medium (37) was inoculated from a fresh Luria-Bertani (47) agar plate and incubated overnight at 30°C and 170 rpm. The cells were harvested and used to inoculate 60 ml of CGXII medium (28) containing 0.03 g of protocatechuic acid/liter and 0.2 M glucose. This second preculture was grown overnight at 30°C and 120 rpm in a 500-ml baffled shake flask. The main culture was inoculated with cells from the second preculture and grown under the same conditions. Strains with the panBC deletion were supplemented with 3 µM sodium D-pantothenate, and strains with the ilvA deletion were supplemented with 3.4 mM L-isoleucine. Strains carrying pJC1-derived plasmids were cultivated in the presence of 50 µg of kanamycin/ml. When desired, branched-chain amino acids were added to CGXII medium before the pH was titrated to 7.0, and then the medium was autoclaved.


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  		TABLE 1. Strains and plasmids

For transcriptome analysis, proteome analysis, and determination of AHAS activity, the CGXII preculture contained the same supplements as the main culture to ensure full adaptation. Main cultures were harvested in the exponential growth phase. Altogether, the bacteria were cultivated for at least 10 generations under equivalent conditions.

Generation of C. glutamicum DNA microarrays.
DNA microarrays based on the PCR products of C. glutamicum genes were used for global gene expression analysis. The genes were amplified in 96-well plates with genomic DNA of C. glutamicum ATCC 13032 as a template and gene-specific primers purchased from degussa (Frankfurt, Germany). The sizes and quantities of the PCR products were checked by gel electrophoresis. Then, the PCR products were precipitated with isopropanol, resuspended in 3x SSC (20x SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), and transferred to 384-well plates as described previously (57, 61; http://cmgm.stanford.edu/pbrown/mguide/index.html). The PCR products were printed onto poly-L-lysine-coated glass slides using an arraying robot (http://cmgm.stanford.edu/pbrown/mguide/index.html). The DNA microarrays were rehydrated in a humidity chamber containing 1x SSC, UV cross-linked (650 µJ), and blocked in 230 ml of methylpyrrolidinone containing 15 ml of 1 M boric acid (titrated to pH 8.0 with sodium hydroxide) and 4.4 g of succinic anhydride (57, 61; http://cmgm.stanford.edu/pbrown/mguide/index.html). Depending on the series, the DNA microarrays contained PCR products for up to 3,530 of 3,567 predicted C. glutamicum open reading frames. Most genes were represented by a single spot, but 506 genes were represented by two spots. Up to 100 spots of C. glutamicum genomic DNA were present as a quality control and for normalization. As negative controls, {lambda} DNA, E. coli genomic DNA, and the E. coli aceK gene were included.

Total RNA preparation and cDNA synthesis.
Aliquots (~25 ml) of exponentially growing C. glutamicum cultures (optical density at 600 nm [OD600], between 3 and 5) were added to 25 g of crushed ice precooled to -20°C and immediately harvested by centrifugation (5 min; 3,500 x g; 4°C) as previously described for E. coli (57). The cells were resuspended in 350 µl of RNeasy RLT buffer (Qiagen, Hilden, Germany) and mechanically disrupted by 30 s of bead beating with 0.5 g of 0.1-mm-diameter zirconium-silica beads (Roth, Karlsruhe, Germany) using a Silamat S5 (Vivadent, Ellwangen, Germany). After centrifugation (2 min; 14,500 x g), the supernatant was processed using the RNeasy system (Qiagen) with DNase on-column treatment according to the manufacturer's instructions for RNA extraction. The quantity and quality of the extracted total RNA were determined by UV spectroscopy (at 260, 280, and 230 nm) and denaturing formaldehyde agarose gel electrophoresis (47).

Identical amounts (20 to 25 µg) of total RNA were used for random hexamer-primed synthesis of fluorescently labeled cDNA by reverse transcription with Superscript II (GibcoBRL-Life Technologies, Gaithersburg, Md.) using the fluorescent nucleotide analogue FluoroLink Cy3-dUTP (green) or Cy5-dUTP (red) (Amersham Pharmacia, Little Chalfont, United Kingdom) as described before (30, 57). The labeled cDNA probes were purified and concentrated using Microcon YM-30 filter units (Millipore, Bedford, Mass.) (30; http://cmgm.stanford.edu/pbrown/mguide/index.html).

DNA microarray hybridization and washing.
Combined Cy5- and Cy3-labeled cDNA probes containing 1.2 µg of poly(A) (Sigma, Taufkirchen, Germany)/µl as a competitor, 30 mM HEPES, and 0.3% sodium dodecyl sulfate (SDS) in 3x SSC, were hybridized to the arrays for 5 to 16 h at 65°C. After hybridization, the arrays were washed in 1x SSC containing 0.03% SDS and finally in 0.05x SSC. The DNA microarrays were dried by brief centrifugation (5 min; 45 x g). For detailed protocols, see reference 30 and http://cmgm.stanford.edu/pbrown/mguide/index.html.

Data normalization and gene expression analysis.
Immediately after stringent washing of the arrays, fluorescence intensities at 635 and 532 nm were acquired using a GenePix 4000 laser scanner (Axon Inc., Union City, Calif.) and processed as TIFF images. Raw fluorescence data were analyzed quantitatively using GenePix version 3.0 software (Axon Inc.). Data were normalized to the average ratio of C. glutamicum genomic DNA. The normalized ratio of the median (GenePix) was taken to reflect the relative RNA abundance for spots whose green or red fluorescence signal was at least threefold above the fluorescence background. When both fluorescence signals were less than threefold above background, the signals were considered too weak to be analyzed quantitatively. For statistical analysis (2, 42), P values from independent replicate experiments were calculated based on Student's t test using log-transformed gene ratios and genomic DNA ratios which were normalized to zero (33). Only genes showing significantly changed RNA levels (P values of <0.05) were considered for further analysis. Analysis of gene expression data was performed by selecting genes showing at least twofold-increased or -decreased average RNA levels. All genes belonging to a putative operon were listed if at least one gene of the operon showed significant expression changes.

Proteome analysis.
Proteome analysis of the soluble protein fraction was performed by two-dimensional (2-D) gel electrophoresis essentially as described previously (48). Cells were cultivated as described above and harvested at an OD600 of 3 to 5 by centrifugation (5 min; 3,500 x g; 4°C). After being washed in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), the cells were resuspended in either 1 or 10 ml of TE buffer containing Complete protease inhibitor (Roche Diagnostics, Mannheim, Germany), 100 µg of RNase A, and 25 µg of DNase I, depending on whether disruption was performed by bead beating (1 ml of cell suspension was added to 1 g of zirconium-silica beads and subjected to four cycles [30 s] of bead beating) or by passage through a French pressure cell (10 ml of cell suspension was passed five times through a French pressure cell [SLM AMINCO Spectronic Instruments, Rochester, N.Y.] at 172 MPa). Intact cells and cell debris were removed by centrifugation (20 min; 27,000 x g; 4°C), and the soluble protein fraction was separated from the membrane protein fraction by ultracentrifugation (1 h; 150,000 x g; 4°C). Protein concentrations were determined using the BC assay kit (Pierce Chemical Company, Rockford, Ill.), and 300 µg of protein was precipitated with acetone. After solubilization, isoelectric focusing was performed using an IPGphor electrophoresis unit (Amersham Pharmacia) and 18-cm-long Immobiline DryStrips (Amersham Pharmacia) with a pH range of 4 to 7 or 4.5 to 5.5. After being focused, the Immobiline DryStrips were equilibrated and 2-D separation was performed using a Multiphor II electrophoresis unit and Excel SDS gradient gels (12 to 14%; Amersham Pharmacia). The gels were fixed, Coomassie stained, destained, and dried as described previously (48).

The gels were scanned with a JX-330 scanner (Sharp, Tokyo, Japan), and the images were analyzed using ProteomeWeaver 2-D gel analysis software version 1.1.3 (Definiens Imaging GmbH, Munich, Germany). Spots of interest were excised and digested with trypsin, and the peptide masses were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as described previously (48). Peptide mass lists were used to search a tryptic digest database of 3,746 C. glutamicum proteins provided by degussa.

Determination of amino acid concentrations by HPLC.
L-Valine concentrations were determined by automatic precolumn derivatization with ortho-phthaldialdehyde (35) and reversed-phase high-performance liquid chromatography (HPLC) (HP1100 series; Hewlett-Packard, Waldbronn, Germany) with fluorimetric detection (excitation at 230 nm; emission at 450 nm). Hypersil ODS 5-µm columns were used (precolumn, 40 by 4 mm; column, 120 by 4 mm; Chromatographie Service GmbH, Langerwehe, Germany). The buffer gradient consisted of 0.1 M sodium acetate, pH 7.2 (with 0.03% sodium azide), as the polar phase and methanol as the nonpolar phase. Quantification was performed with L-asparagine as an internal standard and by comparison of the sample peaks with an external standard.

Determination of AHAS activity.
Cells (50 ml) were harvested at an OD600 of 3 to 5 by centrifugation, washed with 50 ml of 100 mM potassium phosphate buffer (pH 7.5), and suspended in 1 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 26% (vol/vol) glycerol. The cells were disrupted by sonification with a microtip-equipped Branson Sonifier W-250 (Branson-Emerson, Danbury, Conn.) for 10 min on ice (intensity, 20%; pulse length, 20%). Cell debris and intact cells were separated from the cell extract by centrifugation (1 h; 14,500 x g; 4°C). The enzyme assay was performed as described previously (16) for 15 min at 30°C. Acetolactate formed from pyruvate was decarboxylated to acetoin and detected by the colorimetric method of Westerfeld (58), which is based on a reaction between acetoin and the guanidino group of creatine in the presence of {alpha}-naphthol and alkali. The absorbance at 530 nm was compared with an acetoin standard. Protein concentrations were determined using the biuret method (22). Specific AHAS activities are given as mU per milligram (nanomoles of product formed per minute and milligram of protein).

Determination of ornithine carbamoyltransferase activity.
Cultivation and crude extract preparation were performed as described for the AHAS activity measurement. The reaction mixture contained cell extract, 15 mM L-ornithine, and 10 mM lithium carbamoylphosphate in potassium phosphate buffer (100 mM; pH 7.5). The enzyme assay was performed for 15 min at 30°C and stopped by addition of HCl to a concentration of 0.2 M. The L-citrulline formed during the reaction was detected by reversed-phase HPLC with an L-citrulline standard as described for the determination of amino acid concentrations.


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RESULTS
 
Growth of wild-type C. glutamicum and the VAL1 strain in the presence of valine.
Since the presence of 30 mM valine inhibited the growth of the lysine producer C. glutamicum MH20-22B (18), we were interested in examining the effects on the growth of the C. glutamicum wild-type strain ATCC 13032 and the valine production strain VAL1. Cells were cultivated in CGXII minimal medium with 0.2 M glucose and different valine concentrations, and the growth rates were determined. The medium for strain VAL1 was supplemented with 3 µM sodium D-pantothenate and 3.4 mM L-isoleucine. As shown in Fig. 2, L-valine at concentrations up to 200 mM had no effect on the growth rate of the wild type. In contrast, the VAL1 strain derived from this wild type showed decreasing growth rates at increasing L-valine concentrations. Half-maximal inhibition was found at a concentration of 250 mM L-valine (data not shown). These results indicate that the effect of external L-valine on the growth of C. glutamicum is strain specific. In the context of these studies, it was also shown that wild type C. glutamicum is unable to use L-valine as a sole carbon or a sole nitrogen source (data not shown). This clearly indicates that C. glutamicum lacks one or several enzyme activities required for L-valine degradation.



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  		FIG. 2. Effect of L-valine in the culture medium on growth rates (µ) of wild-type C. glutamicum, ATCC 13032 ({circ}), and the valine production strain, VAL1 ({blacksquare}). The average growth rates and standard deviations of at least two independent cultivations are given.

Influence of L-valine on global gene expression in wild type C. glutamicum and the VAL1 strain.
In order to find the cause of the different effects of valine on growth of the wild type and the VAL1 strain, its influence on global gene expression was studied by transcriptome analysis. In the case of the wild type, parallel cultures were grown in CGXII glucose minimal medium either with or without 50, 100, or 300 mM L-valine. The cells were kept in the exponential growth phase by repeated dilution and harvested for RNA extraction after at least 10 generations. Relative RNA levels were determined by hybridization to DNA microarrays representing up to 3,530 different open reading frames (ORFs) of C. glutamicum ATCC 13032 (see Materials and Methods). Table 2 lists statistically significant gene expression changes (P value, <0.05) of 23 ORFs with at least twofold-decreased RNA levels and 16 genes with at least twofold-increased RNA levels (in all cases, the RNA levels represent the ratio with valine/without valine). Additionally, genes that showed changes of less than twofold but that formed an operon with one of the 39 genes that had at least twofold-altered mRNA levels were included.


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  		TABLE 2. ORFs showing altered relative mRNA levels in response to L-valine in the wild-type C. glutamicum ATCC 13032

In the case of the VAL1 strain, parallel cultures were grown in CGXII glucose minimal medium either without L-valine (growth rate, 0.35 h-1) or with 40 mM L-valine (growth rate, 0.29 h-1) and used for RNA isolation and global gene expression analysis as described for the wild type. The use of 40 mM valine was a compromise, because at that concentration the effect of valine on the growth of VAL1 was already clearly detectable but presumably not so strong that all gene expression changes were due to the reduced growth rate. In this context, it has to be mentioned that the VAL1 strain is capable of producing ~90 mM L-valine by itself. However, due to the dilution steps, there was <5 mM valine produced by VAL1 in the medium at the time of RNA isolation. As summarized in Table 3, 11 and 10 ORFs showed significantly changed RNA levels (P < 0.05) that were at least twofold decreased or increased, respectively, after growth in the presence of valine. Table 3 also includes those genes whose RNA levels changed less than twofold but which presumably are cotranscribed with one of the 21 genes showing at least twofold changes.


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  		TABLE 3. ORFs showing altered relative mRNA levels in response to L-valine in the valine production strain VAL1a

A comparison of the gene expression changes upon valine addition in the wild type and the VAL1 strain identified common as well as strain-specific expression changes. Three genes showed increased expression in the presence of valine, i.e., leuD, ileS, and its adjacent ORF (Tables 2 and 3). The leuD gene encodes the small subunit of isopropylmalate dehydratase, an enzyme involved in the biosynthesis of L-leucine from 2-oxoisovalerate (39). The levels of RNA of leuC (ORF 2737; NCgl1262), encoding the large subunit of this enzyme, were also increased in both strains but showed P values of >0.05. The ileS gene encodes isoleucyl tRNA synthetase, i.e., the enzyme that charges isoleucine tRNA.

Expression of several genes or operons, e.g., that of the prpD2B2C2 operon, which is essential for growth on propionate (10) and encodes enzymes involved in the methylcitrate cycle, i.e., 2-methylcitrate synthase (prpC), 2-methylcitrate dehydratase (prpD), and 2-methylisocitrate lyase (prpB), was increased in the presence of valine only in the wild type (Tables 2 and 3). Expression of the homologous prpD1B1C1 operon, which is not essential for growth on propionate (10), was also increased. Similarly, increased expression was found for the putative narKGHJI operon that encodes a nitrate-nitrite transport protein and the four subunits of nitrate reductase (narG, with a ratio of 2.0 and a P value of 0.07) and for nearly all genes involved in arginine biosynthesis (argC, argB, argD, argF, argG, argH, and argR) (8, 9, 46). The increased RNA level of the argF gene during growth in the presence of valine correlated with increased activities of ornithine carbamoyltransferase (the argF gene product). Wild-type cells grown in the presence or absence of valine had ornithine carbamoyltransferase activities of 210 and 95 mU/mg of protein, respectively. In the VAL1 strain, the ornithine carbamoyltransferase activities were 145 and 120 mU/mg of protein in the presence or absence of valine, respectively.

Expression of the putative oppABCD operon encoding an oligopeptide ABC transport system was significantly increased only in strain VAL1 (Tables 2 and 3). Finally, it was obvious that the levels of mRNA of ilvBN, which encodes AHAS, were increased in the presence of valine only in the VAL1 strain but decreased or almost unaltered in the wild type (Tables 2 and 3).

Influence of L-valine on the protein profile of wild-type C. glutamicum and the VAL1 strain.
The influence of valine in the growth medium on protein abundances in the wild type and the VAL1 strain was examined using 2-D gels. The strains were cultivated in CGXII glucose minimal medium with or without 300 mM L-valine in the case of the wild type and with or without 40 mM L-valine in the case of the VAL1 strain. After growth for at least 10 generations, the cells were harvested in the exponential phase, and crude extracts were prepared. For each of the four different conditions, three independent cultivations were performed and used for protein profiling. For each of the 12 samples, 2-D gel electrophoresis was carried out both in a pH range of 4 to 7 and in a range of 4.5 to 5.5. The Coomassie-stained gels were analyzed quantitatively using ProteomeWeaver software. Tables 4 and 5 summarize those proteins that showed at least twofold changes in abundance due to the presence of valine. Because of higher standard deviations observed between protein spot intensities in comparison to the DNA microarray results, the criterion for significant changes was set to a P value of <0.1 in Student's t test (in comparison to a P value of <0.05 for the relative RNA levels).


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  		TABLE 4. Proteins showing altered abundances on 2-D gels in response to L-valine in the wild-type C. glutamicum ATCC 13032


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  		TABLE 5. Proteins showing altered abundances on 2-D gels in response to L-valine in the valine production strain VAL1a

In the wild type (Table 4), 11 proteins showed decreased abundance in response to the presence of valine. However, none of the corresponding genes displayed altered relative RNA levels (all corresponding spots could be evaluated and showed ratios of ~1). Thus, the decrease in protein concentration should be due to regulation at the level of translation or protein stability rather than to transcriptional regulation. Three proteins showed increased abundance in response to valine, which in all cases correlated with increased RNA levels. The 2-methylcitrate dehydratase PrpD2 had an eightfold-increased level (mRNA level, sixfold), the arginine repressor ArgR had a fivefold-increased level (mRNA level, twofold), and N-acetylglutamate semialdehyde dehydrogenase, ArgC, had a fourfold-increased level (mRNA level, twofold).

In the VAL1 strain, two proteins showed reduced abundance in response to valine, one of which was the thiamine diphosphate-dependent pyruvate dehydrogenase (EC 1.2.4.1; component E1), which is encoded by aceE and is part of the pyruvate dehydrogenase complex (Table 5). The corresponding genes showed unaltered RNA levels under the same conditions (data not shown). Three proteins displayed increased levels in response to valine, i.e., the translation elongation factor EF-G (twofold increase); PurH (phosphoribosylaminoimidazolecarboxamide formyltransferase-IMP cyclohydrolase), a bifunctional enzyme involved in purine biosynthesis (fourfold increase); and IlvB, the large subunit of AHAS. Whereas the genes encoding EF-G and PurH showed no changes in the RNA level in response to valine, the ilvB mRNA level was increased twofold in the presence of valine (Table 3). Remarkably, eight different spots were identified as IlvB, all having quite similar masses of ~64 to 67 kDa but pIs ranging from ~4.7 to 5.4. This could be explained by successive degradation of the C terminus (http://www.expasy.org/tools/pi_tool.html). The 30 C-terminal amino acids include 10 aspartate and glutamate residues, and their successive degradation would cause predicted shifts in pI from 4.82 to 5.14 and in mass from 66.8 to 63.8 kDa.

Influence of valine on the AHAS activity of the wild type and the VAL1 strain.
The transcriptome studies revealed different effects of valine on the ilvBN mRNA levels in the wild type and the VAL1 strain, which at least partly correlated with the protein levels. Since AHAS is the key enzyme of branched-chain amino acid biosynthesis, it was important to complement the mRNA and protein data by activity measurements. As shown in Table 6, the AHAS activity in the wild type (20 mU/mg of protein) was not influenced by the presence of 300 mM valine in the growth medium. In the VAL1 strain, the activity of cells grown in the absence of externally added valine was 200 mU/mg of protein. This 10-fold increase compared to the wild type is most probably due to increased expression of ilvBN from the plasmid pJC1ilvBNCD. After growth in the presence of 40 mM added valine, the AHAS activity rose to 700 mU/mg. This 3.5-fold increase in AHAS activity correlated well with the 2.5-fold-increased RNA levels of the ilvBN genes and with the increased IlvB protein level.


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  		TABLE 6. AHAS activity after cultivation of wild-type C. glutamicum ATCC 13032 and of the valine production strain VAL1 in the presence or absence of L-valine

Influence of branched-chain amino acids on growth and AHAS activity of the VAL1 strain.
To examine whether the inhibitory effect of valine on growth and the stimulatory effect on AHAS activity of strain VAL1 are specific for valine and to gain more insight into the mechanism causing these effects, the influences of L-leucine and L-isoleucine and of all possible combinations of branched-chain amino acids (40 mM each) on growth and on AHAS activity were tested. The doubling times of the different cultures are shown in Fig. 3A, and the corresponding AHAS activities are shown in Fig. 3B. It is obvious that not only valine but also leucine inhibited the growth of VAL1 and stimulated AHAS activity. From the effects of the different combinations, it is very clear that only the mixture of valine and leucine had qualitatively the same effect as valine or leucine alone, whereas all combinations that included 40 mM isoleucine had no effect on either growth or AHAS activity. Thus, the presence of 40 mM isoleucine (instead of 3.4 mM, always present as a supplement) was able to abolish the effects of valine and leucine. In contrast to the VAL1 strain, the wild type transformed with plasmid pJC1 showed doubling times of 1.6 to 2.1 h under all conditions mentioned above, slightly increased AHAS activity upon isoleucine addition (120% of the activity without isoleucine), and decreased activities in response to all other conditions mentioned (40 to 70% of the activity obtained without amino acid addition).



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  		FIG. 3. Effects of L-valine, L-leucine, and L-isoleucine (40 mM each) addition to the culture medium on doubling time (A) and specific AHAS activity (B) of C. glutamicum VAL1. The error bars indicate standard deviations.

Correlation between valine inhibition and the ilvA deletion of strain VAL1.
The results described above clearly indicated that the effects of valine and leucine on the growth and AHAS activity of the VAL1 strain were caused by a limitation of isoleucine, which is required by this strain as a supplement due to the deletion of the ilvA gene encoding threonine dehydratase. To confirm this conclusion, the effect of 200 mM valine on the growth of a number of isogenic derivatives of the wild type, ATCC 13032, was tested. As shown in Fig. 4, all six strains investigated had the same doubling time (Td) of ~1.7 h in the absence of valine. In the presence of valine, there was a slight increase in the doubling time due to the presence of plasmid pJC1ilvBNCD (Td, ~2 h), but a large increase was due to the ilvA deletion (Td, ~2.7 h). The combination of plasmid pJC1ilvBNCD and the ilvA deletion led to an even greater increase of the doubling time (Td, ~3.5 h), indicating that these effects were synergistic. Strains carrying the plasmid pJC1ilvBNCD produced more valine than the respective pJC1 control strains and thus were exposed to higher valine concentrations. The deletion of the panBC genes had no effect on growth in the presence of valine. These data clearly support the assumption that isoleucine limitation is responsible for growth inhibition by valine and leucine in the VAL1 strain.



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  		FIG. 4. Doubling times of different strains derived from C. glutamicum ATCC 13032 without (shaded bars) or with (solid bars) addition of 200 mM L-valine. The strains utilized were ATCC 13032 (a), 13032{Delta}panBC (b), 13032(pJC1ilvBNCD) (c), 13032{Delta}ilvA (d), 13032{Delta}ilvA(pJC1ilvBNCD) (e), and VAL1 [13032 {Delta}ilvA {Delta}panBC(pJC1ilvBNCD)] (f). Below the columns, it is indicated if the strains do ({Delta}) or do not (wt) carry a deletion of ilvA or panBC and if the strains carry plasmid pJC1ilvBNCD (+) or not (-).

Abolishment of valine-triggered growth inhibition by supplementation of the VAL1 strain with isoleucyl-isoleucine dipeptide.
From the sum of the results described above, the conclusion was drawn that the isoleucine limitation in the presence of valine or leucine is caused by the competition of these amino acids with isoleucine uptake by the carrier protein BrnQ (15, 52). This carrier is responsible for the uptake of all three branched-chain amino acids, and therefore, high concentrations of valine or leucine compete for the uptake of the essential supplement isoleucine, which usually was present at an initial concentration of 3.4 mM in the medium. Consequently, the inhibitory effects of valine and leucine can be abolished by increased isoleucine concentrations (Fig. 3). Final proof of this explanation was obtained from an experiment in which the dipeptide isoleucyl-isoleucine was used instead of isoleucine as a supplement. In contrast to isoleucine, the dipeptide should not be taken up by BrnQ but by a dipeptide transport system, as in E. coli (12, 51), or by one of the peptide transport systems known from many other organisms (40). The putative OppABCD ABC transport system induced by valine in VAL1 (Table 3) encodes a homologue of a peptide transport system and thus could be induced because of the isoleucine limitation. As expected, growth of the VAL1 strain in the presence of 1.7 mM isoleucyl-isoleucine was not inhibited by valine concentrations of up to 200 mM, whereas the control supplemented with 3.4 mM isoleucine was strongly inhibited (Fig. 5).



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  		FIG. 5. Effects of different L-valine concentrations in the culture medium on growth rates (µ) of the C. glutamicum valine production strain VAL1 when supplemented with 3.4 mM isoleucine ({blacksquare}) or 1.7 mM isoleucine dipeptide ({circ}). The average growth rates and standard deviations of two independent cultivations are given.

As indicated in the introduction, the starting point of this study was the inhibitory effect of valine on growth of the leucine-auxotrophic lysine production strain C. glutamicum MH20-22B (18). This effect could now also be explained by competition of valine for leucine uptake via BrnQ. Growth experiments confirmed that the inhibitory effect of valine was abolished when leucine was provided in the form of the dipeptide alanyl-leucine instead of leucine (data not shown).

Influence of externally added valine on valine production by the VAL1 strain.
Since valine inhibited the growth and stimulated the AHAS activity of the VAL1 strain supplemented with isoleucine, it was interesting to examine the influence of externally added valine on valine production. The strain was cultivated three times independently with either 0, 40, or 175 mM valine, and the valine concentration was determined after 24, 48, and 72 h. After 48 h, no further increase occurred. As shown in Table 7, the initial addition of valine to the medium had a positive effect on valine production, leading to an increase of 33% (by addition of 40 mM valine) or even 50% (by addition of 175 mM valine).


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  		TABLE 7. Effects of different starting concentrations of L-valine in the medium on L-valine production by VAL1a


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DISCUSSION
 
Based on the observation that L-valine inhibited the growth of the lysine production strain C. glutamicum MH20-22B, the effects of valine on the C. glutamicum wild type, ATCC 13032, and a derived valine producer strain, VAL1, were studied in this work. Remarkably, the growth of the wild type was not influenced by valine, whereas growth of the VAL1 strain was inhibited. Using a variety of techniques, such as transcript profiling, proteome analysis, and enzymatic studies, we could finally ascribe the inhibitory effect of valine on the isoleucine-auxotrophic VAL1 strain to a competition of isoleucine with valine for uptake by BrnQ. This secondary carrier is responsible for the uptake of all branched-chain amino acids in C. glutamicum (52) and has only slightly different affinities for them (15). The isoleucine transport inhibition could be autoamplified by decreased BrnQ levels, since it has been reported that brnQ is expressed only if the internal isoleucine concentration exceeds 0.5 mM (5).

The first and most important hint of the occurrence of a limitation of either isoleucine or leucine in the VAL1 strain was obtained by transcriptome studies showing that the ilvBN mRNA level was increased by valine in the VAL1 strain but not in the wild type (the different levels of ilvB, ilvN, and ilvC RNAs are caused by the fact that three different transcripts are formed, i.e., the full-length ilvBNC transcript, an ilvNC transcript, and an ilvC transcript [28]). This is surprising, as valine was previously shown to cause decreased ilvBNC transcription due to an attenuation mechanism (38). The increased RNA level correlated with an increased IlvB protein level (IlvN was not present on the 2-D gels, which covered pH 4 to 7, due to its predicted pI of 9.15 [http://www.expasy.org/tools/pi_tool.html]) and an increased AHAS activity in the VAL1 strain. Growth studies with all branched-chain amino acids used singly or in combination then revealed that L-leucine had the same effect as L-valine and that this effect could be abolished by higher concentrations of L-isoleucine. Isoleucine, on the other hand, had no effect on growth or AHAS activity. Thus, the inhibitory effect was shown to be due to isoleucine limitation, and this was confirmed by the fact that valine inhibition was caused primarily by the ilvA deletion of the VAL1 strain. Final proof of transport competition between valine and isoleucine was obtained by a growth experiment using 1.7 mM isoleucyl-isoleucine as a supplement instead of 3.4 mM isoleucine. This dipeptide completely abolished valine-dependent growth inhibition, because it is taken up by a peptide transport system rather than by BrnQ and therefore valine does not compete with its uptake.

Besides showing the differential effects of valine on ilvBN expression in the wild type and the VAL1 strain, the transcriptome studies identified totals of 39 and 21 ORFs that showed at least twofold-changed mRNA levels in response to valine in the wild type and the VAL1 strain, respectively. Three ORFs showed similar increases of RNA levels in both strains, i.e., a putative isoleucine-tRNA ligase gene (ileS), an adjacent short hypothetical ORF (186 bp), and the leuD gene. In view of the fact that isoleucine-tRNA ligase from E. coli is derepressed under isoleucine starvation (23), the same could hold true in C. glutamicum. Whereas isoleucine limitation in the VAL1 strain has been unequivocally demonstrated and explained (see above), its occurrence and the reason for its occurrence in the wild type are not clear. One cause could be increased synthesis of the branched-chain amino acid exporter BrnEF (29). Alternatively, a shortage of charged isoleucyl-tRNA could be caused by a competition of valine and isoleucine for binding to isoleucyl-tRNA, as the isoleucyl-tRNA synthetase of E. coli also misactivates valine (49). The leuD gene encodes one subunit of isopropylmalate dehydratase, an enzyme involved in leucine biosynthesis, and the increased leuD mRNA level in the presence of valine might indicate starvation for this amino acid, similar to the case of ileS. The gene for the second subunit of isopropylmalate dehydratase, leuC, showed 6.6- and 1.7-fold-increased RNA levels in the wild type and the VAL1 strain, respectively, but the P values were above 0.05. In the wild type, leucine limitation again could be caused by an increased level of the exporter BrnEF. In the VAL1 strain, however, the reason for a possible leucine limitation is not obvious. Eventually, valine could have a direct effect on leuCD expression in C. glutamicum.

Interestingly, levels of RNAs of the prpD2B2C2 operon and the prpC1 gene of the prpD1B1C1 operon were four- to sixfold increased in the wild type but unchanged or even reduced in the VAL1 strain. In the case of PrpD2, the increased RNA level correlated with an eightfold-increased protein level on 2-D gels. The spots for PrpB2, PrpC2, and PrpC1 could not be detected on the gels. The prpD2B2C2 operon is essential for propionate utilization by C. glutamicum and is induced by propionate (10). The operon shows sequence similarity to the prp operons of Salmonella enterica serovar Typhimurium and E. coli, in which it was shown that the encoded proteins convert propionyl-CoA to pyruvate (26, 53). Since the degradation of valine leads to the formation of propionyl-CoA (36, 60), the induction of the prp genes by valine could indicate involvement in the conversion of valine-derived propionyl-CoA to pyruvate. However, we could show that C. glutamicum is unable to grow with L-valine as a sole carbon source or as a sole nitrogen source. Therefore, induction of the enzymes of the methylcitrate cycle for the purpose of valine degradation does not make sense. One could argue that the ability to catabolize valine was lost fairly recently, whereas the regulatory mechanisms involved remain unchanged. This appears to be unlikely, because isoleucine, which, like valine, can be catabolized via propionyl-CoA and therefore should also induce the prp genes, was found to inhibit expression of the prpD2B2C2 operon (data not shown). In conclusion, the reason for the induction of the prp genes by valine (and their repression by isoleucine) remains unclear. The same holds true for the result showing that valine did not induce the prp genes in the VAL1 strain. The argument that valine induction and isoleucine repression counterbalance each other is unlikely because isoleucine was shown to be limiting.

A comparison of the results of transcriptome and proteome analyses revealed that 13 proteins had reduced levels in the presence of valine, but none of the corresponding genes showed decreased RNA levels. This might be explained by some kind of technical limitation and/or by the fact that a significant part of the regulation of protein synthesis does not occur at the RNA level but by other means, e.g., translational regulation or protein stability. In the cases of the proteins with increased abundance in the presence of valine, most of the corresponding genes (except those for PurH and EF-G) also showed increased mRNA levels.

An important result of our studies was the observation that the presence of valine in the medium stimulates its own production by the VAL1 strain. This effect is presumably due to the increased AHAS activity and to the growth inhibition, which may favor valine production at the expense of biomass formation. These aspects certainly should be considered in the further improvement of VAL1 strain valine production by metabolic engineering.


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ACKNOWLEDGMENTS
 
We thank E. Radmacher, K. Krumbach, and L. Eggeling for kindly providing strains used in this study, T. Polen and G. Sindelar for help with DNA microarrays, and S. Schaffer for assistance with proteome analysis. We are grateful to degussa for making DNA sequences available.

This work was supported by the European Union within the framework of the VALPAN project (QLK 3-2000-00497).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany. Phone: 49 2461 61 5169. Fax: 49 2461 61 2710. E-mail: v.wendisch{at}fz-juelich.de. Back


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Applied and Environmental Microbiology, May 2003, p. 2521-2532, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2521-2532.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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