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Applied and Environmental Microbiology, May 2005, p. 2391-2402, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2391-2402.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Adaptation of Corynebacterium glutamicum to Ammonium Limitation: a Global Analysis Using Transcriptome and Proteome Techniques{dagger}

Maike Silberbach,1 Mathias Schäfer,2 Andrea T. Hüser,3 Jörn Kalinowski,3 Alfred Pühler,4 Reinhard Krämer,1 and Andreas Burkovski1*

Institut für Biochemie der Universität zu Köln, Zülpicher Strasse 47, D-50674 Köln, Germany,1 Institut für Organische Chemie der Universität zu Köln, Greinstrasse 4, D-50939 Köln, Germany,2 Institut für Genomforschung, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany,3 Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany4

Received 22 September 2004/ Accepted 1 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for FoF1-ATP synthase subunits.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Corynebacterium glutamicum was isolated in 1957 by Kinoshita and coworkers in a screening program for L-glutamate-producing bacteria from a soil sample collected at Ueno Zoo in Tokyo, and at that time it was designated as Micrococcus glutamicus (10, 21, 33). Less than 50 years later, enormous amounts of L-glutamate (more than 1,500,000 tons per year) and L-lysine (more than 560,000 tons per year) are produced by use of different C. glutamicum strains, in addition to smaller amounts of some less industrially important amino acids (L-alanine, L-isoleucine, and L-proline) and in addition to different nucleotides (15). In contrast to closely related pathogenic species, such as Corynebacterium diphtheriae, Mycobacterium leprae, and Mycobacterium tuberculosis, C. glutamicum is generally recognized as a nonhazardous organism which is safe to handle. Furthermore, based on its extremely well-investigated central metabolism and well-established molecular biology tools, C. glutamicum is suitable as a model organism for high G+C content gram-positive bacteria in general and mycolic-acid-containing Actinomycetales in particular.

The regulation of nitrogen metabolism in the Actinomycetales was the subject of research mainly in the last few years (for a review, see references 7 and 8). For C. glutamicum, detailed information of transport and assimilation of nitrogen sources as well as nitrogen regulation is available on a molecular level (for a review, see references 7 and 8). Uptake systems for ammonium, creatinine, and glutamate were studied, and assimilatory enzymes and pathways were investigated. Additionally, the key components of nitrogen control were identified; namely, AmtR, the master regulator of nitrogen control in C. glutamicum, GlnK, the sole PII-type signal transduction protein in this organism, and two modifying enzymes, a putative adenylyltransferase and the GlnD protein (32).

While previous studies focused on specific genes or enzymes, here we present a global analysis of the C. glutamicum ammonium limitation response by transcriptional profiling and two-dimensional gel electrophoresis. In this study these two global approaches were combined with the continuous cultivation of cells. In contrast to shaking flask experiments, this technique allows us to establish highly defined growth conditions over a long period of time, i.e., over days or weeks. As a consequence, cells which are optimally adapted to a specific environment or nutrient supply can be investigated.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth.
C. glutamicum type strain ATCC 13032 (1) was grown at 30°C in a modified MM1 minimal medium (20). For continuous, ammonium-limited fermentations the concentration of nitrogen sources in the starting medium and in the substrate feed was decreased compared to the original medium. Urea was omitted from both media, and in the substrate feed ammonium sulfate was reduced from 5 g liter–1 to 2.75 g liter–1. In addition, the glucose content was reduced from 40 g liter–1 to 20 g liter–1. The process was started in the batch mode by inoculation of 1.3 liters starting medium to an optical density at 600 nm (OD600) of 1 using a minimal medium overnight culture. The fermentation was carried out at 30°C and an aeration rate of 1 vvm (volume per volume and min). The pH was automatically adjusted to 7.0 with 3 M NaOH. During the batch phase, the stirrer speed was increased from an initial 600 rpm to a final value of 1,000 rpm. At an OD600 of 18, 30 ml cell broth was harvested for proteome analysis and 1 ml for transcriptome analysis. Then the process was switched to continuous fermentation mode by starting the steady supplementation of substrate feed with a dilution rate of 0.075 h–1. To guarantee a constant filling volume during the whole fermentation, the surplus of culture broth was removed. After the steady state was reached, samples for transcriptome and proteome analyses were taken (see above) and the dilution rate was increased to a value of 0.15 h–1. After the new steady state was reached, again samples were taken.

Total RNA preparation from C. glutamicum.
The frozen aliquots of C. glutamicum cell material were suspended in 700 µl RA1 buffer (NucleoSpinRNA II kit; Macherey-Nagel, Düren, Germany) and immediately disrupted using glass beads and a Q-BIOgene FastPrep FP120 instrument (Q-BIOgene, Heidelberg, Germany). Disruption was performed by two 30-s cycles at a speed of 6.5 m s–1. After the cell debris was separated, the RNA was isolated using the NucleoSpinRNA II kit following the supplier's recommendations. If necessary, a second DNase digestion was performed with DNase I (Amersham Biosciences, Freiburg, Germany) to completely remove the chromosomal DNA. RNA samples were finally stored at –80°C.

Transcriptome analyses.
For transcriptome analyses, 5 µg of total C. glutamicum RNA was used for cDNA synthesis. During reverse transcription of the RNA, aminoallyl-modified dUTP (aa-dUTP; Sigma-Aldrich, Taufkirchen, Germany) was incorporated to prepare the samples for indirect labeling. Afterwards, Cy3 or Cy5 monofunctional NHS-esters (Amersham Biosciences, Freiburg, Germany) were coupled with the aa-dUTPs, excess NHS-esters were removed, and the samples were purified using a MinElute PCR purification kit (QIAGEN, Hilden, Germany). For detailed information concerning cDNA synthesis and fluorescent labeling see reference 16.For hybridization, Cy3- and Cy5-labeled samples were combined and vacuum dried. Microarray slides covering more than 93% of all C. glutamicum genes in four replicates were prehybridized and prepared as described elsewhere (16). The vacuum-dried sample was suspended in 70 µl DIG-Easy Hyb hybridization solution (Roche Diagnostics, Mannheim, Germany). This mixture was used for hybridization under a coverslip inside an in situ hybridization chamber (TeleChem International, Sunnyvale, CA). After washing and drying the microarrays (16), the signal acquisition was performed with a ScanArray 4000 microarray scanner (Perkin-Elmer, Boston, MA). The Imagene 5.0 software (Biodiscovery, Los Angeles, CA) was used for spot finding, signal-background segmentation, and intensity quantification (16). Ratios were calculated and normalization as well as t test statistics were performed using the EMMA microarray data analyses software (9). All signal intensities with ratios above 1.74 were regarded as significant, if a P value below 0.05 was assigned. For each comparison of interest, two independent experiments were performed.

Protein sample preparation and 2-D PAGE.
C. glutamicum cells were disrupted using glass beads and a Q-BIOgene FastPrep FP120 instrument (Q-BIOgene, Heidelberg, Germany) by lysing the cells four times for 30 s and 6.5 m s–1 in the presence of the proteinase inhibitor Complete as recommended by the supplier (Roche, Basel, Switzerland). Proteins were separated by ultracentrifugation in cytoplasmic and membrane-associated protein fractions (13, 14). In this study, only the cytoplasmic proteins were further analyzed. Protein concentrations were determined with Amido Black (28). For isoelectric focusing, 24-cm precast IPG strips, pI 4 to 7, and an IPGphor isoelectric focusing unit (Amersham Biosiences, Freiburg, Germany) were used as described previously (14). One hundred micrograms of protein was focused for 68,000 V · h in a sample buffer containing 6 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 0.5% Pharmalyte (pH 3 to 10), and 0.4% dithiothreitol. The run for the second dimension was carried out using precast 12 to 14% polyacrylamide linear gradient gels (ExcelGel Gradient XL 12-14; Amersham Biosciences, Freiburg, Germany) in a Multiphor II apparatus as described previously (14). After electrophoresis, two-dimensional (2-D) gels were stained with Coomassie brilliant blue (27). The Coomassie-stained gels were aligned using the Delta2D software, version 3.2 (Decodon, Greifswald, Germany). All samples were separated at least twice by 2-D polyacrylamide gel electrophoresis (PAGE) to minimize irregularities (technical replicates). To validate the results, each comparison of interest was performed using samples from two independent experiments (biological replicates). The Delta2D software (version 3.2) also was used for spot quantification. Proteins were regarded as regulated if (i) the corresponding ratios referring to the relative volumes of the spots were changed more than twofold and if (ii) this regulation pattern was found in all biological and technical replicates. All other proteins were classified as "not regulated."

MALDI-TOF MS.
Protein spots of interest were excised from Coomassie-stained 2-D gels for peptide mass fingerprinting via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). All MALDI-MS experiments were conducted in the reflectron mode (resolution, full width at half maximum of ≥10,000) on a Voyager-DE STR reflectron TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany) equipped with a N2-UV laser (337-nm, 3-ns pulse length). The gel pieces were washed twice for 5 min with 500 µl of 50 mM NH4HCO3 and once for 30 min with 500 µl of 50 mM NH4HCO3 to remove all contaminants. Subsequently, the gel pieces were destained twice for 30 min with 50 mM NH4HCO3 in 50% acetonitrile, shrunken with 100 µl acetonitrile for 5 min, and dried under vacuum for 30 min (Concentrator 5301; Eppendorf, Hamburg, Germany). Tryptic in-gel digestion was started by rehydration of the gel matrix by the addition of 1 to 2 µl of 25 mM NH4HCO3 containing 10 µg/ml trypsin (sequence grade; Promega, Madison, WI). After 30 min, 25 mM NH4HCO3 was added to cover the sample and digestion was continued overnight at room temperature. Another 2 µl of 25 mM NH4HCO3 and the following incubation for 90 min at room temperature were used for additional peptide extraction. This peptide solution (0.5 µl) was mixed with 0.5 µl of 5 mg/ml {alpha}-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid on a standard 100-spot stainless steel sample plate (Applied Biosystems, Darmstadt, Germany). All chemicals were of analytical grade and used as purchased from Fluka (Buchs, Switzerland) and Sigma-Aldrich (Steinheim, Germany). Data acquisition and subsequent analysis was done by Voyager Instrument Control Panel software and Voyager Data Explorer software (version V5.1; Applied Biosystems, Darmstadt, Germany). External mass calibration was done close to each sample spot by using calibration mixtures 1 and 2 of the Sequazyme peptide mass standard kit (Applied Biosystems, Darmstadt, Germany). Samples were analyzed manually in the positive reflector mode with delayed extraction of ions (150 ns), 20 kV acceleration voltage, and 66% acceleration grid voltage.

All database searches were performed using the GPMAW software, version 6.0 (Lighthouse Data, Odense, Denmark). The resulting peptide mass lists were compared with a local in-house database of C. glutamicum proteins (Institut für Genomforschung, Universität Bielefeld). The search criteria were set to a mass accuracy of ≤100 ppm and preferably no and maximally one miscleaved peptide per protein. Proteins were considered as identified when more than 30% amino acid sequence was covered by the identified peptides and four or more peptides matching the search criteria with a deviation of mass accuracy based on an incorrect calibration equation.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ammonium limitation stimulated drastic changes in the expression of 285 genes in C. glutamicum wild-type cells (for a complete list of genes with altered expression depending on ammonium supply, see Table S1 in the supplemental material; for genes discussed in this study, see Table 1). These genes include open reading frames encoding transport proteins, proteins involved in nitrogen metabolism and regulation, energy generation, and protein turnover (Fig. 1). Additionally, 74 genes coding for proteins of unknown function or hypothetical proteins are altered in their transcriptional profile. The latter will not be discussed in the following sections.


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  		TABLE 1. Alterations in transcription in response to long-term ammonium limitationa

 


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  		FIG. 1. Functional categories of genes with altered transcription profiles and their part of total regulated genes. For a complete list of genes with altered expression depending on ammonium supply, see Table S1 in the supplemental material.

 
Transport.
Transport systems play a crucial role for the adaptation of cells to a situation of limiting resources, since uptake of a nutrient into the cell is a prerequisite for its utilization. Drastic changes in the transcription of genes encoding different membrane proteins were observed as a response to ammonium limitation in C. glutamicum. Especially, expression of genes encoding a creatinine permease, the two ammonium transporters AmtA and AmtB, and an ABC-type urea uptake system was strongly enhanced by factors between 25 and 75. Only one other gene, glnK (see below), showed similar increases in transcription. Obviously, scavenging for nutrients is a major response of C. glutamicum to ammonium limitation.

Genes encoding other transport systems were less affected. The transcription of NCgl2051 which codes for the secreted component of an ABC-type amino acid transport system was moderately enhanced by a factor of 1.97. Expression of ptsG, encoding a protein involved in glucose uptake, NCgl1968, coding for a putative di- and tricarboxylate transporter, gntP, encoding a putative gluconate uptake system, and NCgl1915-1918, coding for a putative peptide transporter, was moderately increased in response to ammonium limitation (with factors between approximately 2 and 3). The upregulation of the gluABCD cluster might be attributed to the uptake of carbon sources as well, although glutamate can be utilized as a carbon and a nitrogen source in C. glutamicum, since this gene cluster is also moderately enhanced in expression, while transporters working exclusively for the uptake of nitrogen sources show much higher factors of upregulation (see above).

The transcription of betP and putP which code for compatible solute uptake systems is decreased in response to ammonium limitation. Compatible solutes are accumulated inside the cell during hyperosmotic stress (for a recent review, see reference 23) and cold shock. They do not interfere with the cellular metabolism and cannot serve as a nitrogen or carbon source. Under the osmotically strictly controlled conditions of continuous fermentation, their transport is dispensable. Additionally, two genes coding for putative ribitol-specific transport proteins, rmpA and rmpC, were downregulated.

Nitrogen metabolism and amino acid biosynthesis.
To cope with the situation of ammonium limitation and in order to utilize the alternative nitrogen sources taken up by newly synthesized transport systems, cellular pathways used for ammonium assimilation were rearranged and those for the metabolism of other nitrogen sources were activated. In response to ammonium limitation, an enhanced transcription of the gltBD operon encoding glutamate synthase (GOGAT) and the glnA gene, encoding glutamine synthetase (GS), was observed by DNA microarray analyses. The expression of these genes allows the assimilation of ammonium via the high-affinity GS/GOGAT pathway rather than by the low-affinity glutamate dehydrogenase reaction. Since glnA is the only gene encoding an active glutamine synthetase in C. glutamicum and already shows an expression level in nitrogen-rich medium that is enough to satisfy the cellular glutamine demand (25), transcription of glnA is upregulated by a factor of 5 only. In contrast, gltBD expression, which is almost not detectable when cells are grown in nitrogen-rich minimal medium (2, 31), is increased up to 25-fold. Additionally, an up to 10-fold increase in expression of the urease-encoding genes, the ureABCEFGD operon, supports the utilization of urea as an alternative nitrogen source. This result is also in accord with the recently published regulation of ureABCDEFG expression by the master regulator of nitrogen control AmtR (3). As in the case of glnA, this operon also shows a considerable background expression in nitrogen-rich medium. A drastic increase in transcription upon ammonium limitation is not necessary. The enhanced expression of the codA gene, which codes for creatinine deaminase, allows degradation of creatinine to 1-methylhydantoin and ammonia (5). The latter can then be assimilated to produce glutamate or glutamine.

Besides an increase in transcription, a downregulation of genes was observed as well. Especially genes encoding proteins of the L-aspartate (aspA), L-arginine (argB, argC, argD, argF, argG, argH, argJ, argR, and argS), L-leucine (leuA), L-threonine (thrB), L-lysine (lysA), and aromatic L-amino acids biosynthesis pathways (aroA, aroF, and aroG) showed a decreased expression in ammonium-limited chemostat cultures.

Interestingly, expression of the ddh gene coding for meso-diamonopimelate dehydrogenase is downregulated, while transcription of dapD coding for tetrahydrodipicolinate-N-succinyltransferase is increased. These two enzymes are part of the split diaminopimelate pathway (30, 34), which is important for biosynthesis of cell wall precursors and of L-lysine. Downregulation of ddh expression impairs the low-ammonium-affinity branch of this pathway, while upregulation of dapD transcription increases flux via the high-ammonium-affinity branch, allowing synthesis of the cell wall building block diaminopimelate even under ammonium limitation. To avoid a drain of ammonium into L-lysine biosynthesis, which diverts from this pathway, transcription of the lysA gene encoding diaminopimelate decarboxylase is repressed in response to ammonium limitation.

Carbon and energy metabolism.
Active transport of nitrogen sources and ammonium assimilation via the GS/GOGAT pathway needs a higher amount of energy than passive diffusion of ammonia across the cytoplasmic membrane and assimilation by glutamate dehydrogenase. As a consequence, another group of genes exhibiting increased transcription during ammonium limitation are those encoding proteins involved in energy metabolism. Examples are fda and gap, which code for the glycolysis pathway enzymes fructose-1,6-diphosphate aldolase and glyceraldehyde dehydrogenase. Additionally, transcription of a number of genes encoding respiratory chain components is enhanced. These include the sdhA, sdhB, and sdhCD genes encoding succinate dehydrogenase proteins, ccsB coding for cytochrome c assembly protein, qcrA1 encoding a Rieske Fe-S protein, qcrC coding for cytochrome c, ctaE for cytochrome oxidase subunit III, and ctaD for heme/copper-type cytochrome/quinol oxidase subunit I. Upregulation of expression of these genes is moderate, with factors observed between 2 and 4. This is in accord with factors observed for the increased transcription of carbon source uptake systems.

Genes with downregulated transcription in response to ammonium limitation are aceA and aceB, which code for isocitrate lyase and malate synthase, as well as adhA, coding for alcohol dehydrogenase.

Protein stability and turnover.
The increased transcription of the groEL genes, coding for the GroEL chaperonin, and groES, coding for the GroES co-chaperonin might indicate a trend towards protein stabilization in ammonium-limited fermentation. In accord with this hypothesis, a downregulation of transcription of protease-encoding genes pepB, NCgl0274, NCgl0440, and NCgl2737 was observed.

Cell division and cell wall synthesis.
Since ammonium limitation impairs growth and the growth rate is restricted to values of 0.075 and 0.15 h–1 by the dilution rate of continuous fermentation mode, the cell division machinery of C. glutamicum might be at least partially dispensable under these conditions. We observed that transcription of ftsI, encoding the cell division protein FtsI, wzz, coding for a cell surface polysaccharide biosynthesis protein, wzx, coding for a putative translocase involved in export of a cell surface polysaccharide, mraW, coding for a S-adenosylmethionine-dependent methyltransferase involved in cell envelope biogenesis, and parA2, coding for a putative ATPase involved in chromosome partitioning, was decreased moderately by a factor of approximately 0.5.

Regulatory systems.
Based on the strongest increases in transcription observed in ammonium-limited cells, the most important genes in this category are glnD and glnK, with factors of increase of about 17 and 32. This is in accord with the function of the corresponding gene products, since glnD and glnK encode essential proteins for the C. glutamicum nitrogen starvation response (26).

Besides these drastic changes, moderate alterations (with factors between 0.3 and 3.0) in the transcription of genes coding for various regulators (argR, whiB4, whiB1, NCgl1317, NCgl1856, NCgl1887, NCgl2199, NCgl2684, and NCgl2941) and two different proteins of two-component signal transduction systems (the cgtR3 gene encoding a response regulator and the cgtS10 gene coding for a sensor kinase) were observed (Table 1), indicating a modulation of other metabolic pathways not directly connected to nitrogen metabolism.

Growth rate-dependent effects on transcription.
The chemostat experiments carried out allowed not only insights into ammonium limitation-dependent expression of genes but also the first data on growth rate-dependent transcription. When ammonium-limited fermentations with different dilution rates were compared, several genes were identified that showed a growth rate-dependent expression pattern (Table 2). Transcription of the atpFHAGDC genes encoding ATP synthase Fo subunit b and F1 subunits {delta}, {alpha}, {gamma}, ß, and {varepsilon} as well as expression of the cmt3, cmt4, and cmt5 genes coding for corynomycolyl transferases, the tkt gene coding for transketolase, the galU2 gene for UDP-glucose pyrophosphatase, the pgi gene for glucose-6-phosphate isomerase, cynT encoding carbonic anhydratase, betP coding for an osmoregulated glycinebetaine uptake system, and six genes encoding ribosomal proteins are increased in faster growing cells. In contrast to this upregulated gene expression, transcription of the cysD, cysH, cysI, cysJ, and cysN genes, coding for enzymes involved in sulfur metabolism, the mez gene, coding for malic enzyme, the sigma factor-encoding sigB and sigE genes, as well as clpB, coding for an ATPase with chaperone activity, is decreased in fast-growing cells.


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  		TABLE 2. Growth rate-dependent gene expressiona

 
Proteome analysis.
While transcriptome analyses reveal changes in the amount of mRNA transcribed, proteome analyses allow us to compare the actual amount of proteins present under different environmental conditions. Previously carried out proteome analyses of C. glutamicum shaking flask cultures revealed that differences in the protein profile of cells grown under nitrogen-rich conditions and nitrogen-starved cells are hardly detectable when conventional staining techniques (i.e., Coomassie or silver staining) are used. To circumvent this problem, in different studies in vivo, [35S]methionine labeling and autoradiography have been used to improve the sensitivity of two-dimensional gel electrophoresis (26, 29). Besides these methods relying on the use of radio-nucleotides, the use of continuous fermentation is an interesting alternative. Continuous fermentation offers the possibility to obtain a well-defined and highly reproducible growth state of the cells. The cell population can adjust over a long period of time to a constant environment, which significantly improves the sensitivity of the proteome approach as shown in this study.

Spots detected with different intensities and sizes depending on the nitrogen supply were excised from the gel, and the proteins were identified by peptide mass fingerprint analyses using tryptic in-gel digest and MALDI-TOF MS (Table 3; Fig. 2). Protein spots with increased size on gels loaded with cell extract from nitrogen-deprived cultures were identified as six of the seven urease subunits (the ureA, ureB, ureC, ureE, ureG, and ureD gene products), a putative ornithine cyclodeaminase (encoded by the ocd gene), creatinine deaminase (codA gene product), the GlnK protein, tetrahydrodipicolinate-N-succinyltransferase (encoded by dapD), and the glnA gene product glutamine synthetase. Besides these proteins involved in nitrogen metabolism, proteins of carbon and energy metabolism were found, namely, glyceraldehyde-3-phosphate dehydrogenase (gap gene product) and the ATP synthetase F1 {delta} subunit (encoded by atpH). Additionally, a putative L-2,3-butanediol dehydrogenase (butA) and two hypothetical proteins (NCgl2450 and NCgl2451) were found to be present in higher amounts during nitrogen limitation.


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  		TABLE 3. Proteins identified by 2-D PAGE and peptide mass fingerprint analysisa

 



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  		FIG. 2. Protein profiles of C. glutamicum cells grown with different nitrogen supply. Two-dimensional protein profiles of cytoplasmic proteins of batch (nitrogen-rich) phase (A) and continuous (nitrogen-limited) phase at a low dilution rate (0.075 h–1) (B). One hundred micrograms of protein was separated on each gel; staining was carried out with Coomassie brilliant blue. For each condition, at least two independent gels were analyzed and only spots which showed the same pattern in all gels were subjected to MALDI-TOF MS. Positions of molecular weight markers and pH values are indicated. Numbers indicate the positions of proteins in Table 3.

 
Proteins with decreased concentration in response to nitrogen starvation were 2-isopropylmalate synthase (leuA), glutamate N-acetyltransferase (argJ), and sulfate adenylate transferase subunit 2 (cysD), which are involved in the biosynthesis of different amino acids.

An interesting aspect of proteome analyses is the possibility to identify protein modifications. In this study, we were able to identify the modification of the central nitrogen signal transduction protein GlnK, which is an AMP group protein (32). Spot 65 corresponds to the unmodified GlnK protein, while the protein of spot 66 carries an adenylylation as indicated by MALDI-TOF MS (data not shown). Additionally, a pI shift of glutamine synthesis from approximately 4.6 to 5.1 was observed when cells were grown under nitrogen limitation. This is in accord with a deadenylylation of the GS enzyme in response to nitrogen deprivation, which was reported previously (17, 18).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcriptome analyses by DNA microarrays and proteome analyses allow a global view of the responses of a cell induced by changes in environmental conditions. In this study, the response of the biotechnologically important mycolic acid-containing actinomycete C. glutamicum to ammonium limitation was tested in chemostat experiments. A major advantage of the cultivation in continuous fermentation mode is the possibility to establish highly defined long-term conditions of nutrient limitation, while in shaking flasks only a nutrient starvation can be initiated by a total lack of nutrients. Moreover, by comparing different dilution rates in ammonium-limited chemostats, we were able to distinguish between specific and general cellular responses to ammonium limitation and to separate these from growth rate-dependent regulatory mechanisms.

The core response of ammonium-limited cells is characterized by the highest factors of increases in transcription. Our transcriptome analyses indicated that scavenging of nitrogen sources, an adaptation strategy which was first observed for the gram-negative model organism Escherichia coli (36), is a major response to nitrogen starvation in the gram-positive C. glutamicum as well. In this bacterium, synthesis of new transport systems includes ammonium uptake systems, a creatinine permease, an uptake system for urea, and a putative peptide transport system.

An adaptation of similar importance might be the rearrangement of metabolic pathways. As indicated by our transcriptome data, the GS/GOGAT pathway is recruited for ammonium assimilation during transcription. Interestingly, the factor of upregulation of gltBD expression observed in this study is much higher than previously reported (31). This result indicates an extremely high sensitivity of the DNA microarray approach applied here. This view is supported by the fact that the ureABCEFGD cluster was found to be highly upregulated as well. Most likely due to low mRNA abundance, a transcriptional upregulation of these genes was not detected previously although urease activity increased significantly in response to nitrogen limitation in shaking flask experiments (24) and only very recently the mechanism of transcription control of the urease-encoding genes has been solved (3).

Besides the need for sufficient nitrogen sources, a bacterial cell is challenged by an extra demand of energy when subjected to nitrogen limitation. It has already been published that C. glutamicum has an extremely high energy demand in case of a complete fixation of ammonium via the GS/GOGAT pathway in comparison for example to E. coli (29). These calculations were based on the nitrogen-assimilating enzymes only and did not include the extra demand of energy necessary for enhanced transport processes or synthesis of new proteins. As a consequence, increasing the amount of glycolytic enzymes and redox chain components as indicated by the transcriptome analyses is a reasonable response to nitrogen starvation. The results obtained by transcriptome analyses are supported by the experiments of Schmid and coworkers (29) who observed an enhanced oxygen consumption of cells deprived of nitrogen sources. In contrast to this situation in C. glutamicum, a decreased respiration rate during nutrient starvation was observed for the closely related pathogen M. tuberculosis (6). However, in this case cells were suspended in phosphate-buffered saline and therefore deprived of nitrogen and carbon sources as well as various other nutrients. In addition to the changes in expression of genes encoding glycolytic enzymes and respiratory chain components, carbon uptake systems were induced. The factors of upregulation observed for the increase in transcription of genes encoding components of carbon metabolism and respiration were quite moderate, indicating a more general or indirect response to the situation of ammonium limitation.

During nitrogen limitation in continuous fermentation mode, the growth rate is restricted to very low values. The decreased transcription of two genes encoding proteins with a function in cell division as well as three genes coding for cell envelope biosynthesis proteins indicated a reduction of the cell division machinery of C. glutamicum. Additionally, the overall protein stability was increased by positive regulation of three chaperonines on the one hand and the decreased expression of five different protease- and peptidase-encoding genes.

The comparison of different dilution rates in ammonium-limited chemostats allowed us to detect even more indirect effects. Some genes, e.g., those encoding ATP synthase subunits, we regulated in parallel to the growth rate of C. glutamicum, which is of course also influenced by nitrogen availability but also by other factors (trace elements, temperature, etc.). In shaking flasks experiments, it is impossible to distinguish between specific or indirect effects of nitrogen limitation, since a starvation can only be realized by complete lack of any nitrogen source.

The transcriptome approach used in this study was combined with and validated by proteome analyses, a combination used by other groups for different bacteria (11, 12, 22, 35) as well. The lack of a suitable method for the two-dimensional gel electrophoresis of membrane proteins with more than one transmembrane helix is a clear drawback of proteome analyses. This is also the reason why only cytoplasmic proteins were analyzed by 2-D PAGE in this study although a considerable number of transporter-encoding genes were identified by transcriptome analyses. However, the advantage of this combination results from the fact that proteome techniques allow us to analyze not only the synthesis but also the modification of proteins (4), which might be of major importance for regulatory networks. As shown in this study, the usefulness of these global analysis techniques can be further improved by their combination with the use of continuous cultivation techniques. The longer adaptation time for the cells allows a better analysis of differences in protein profiles.


   ACKNOWLEDGMENTS
 
This work was supported by the Bundesministerium für Forschung und Technologie ("Neue Methoden zur Proteomanalyse" and "GenoMik" programs) and by the Deutsche Forschungsgemeinschaft (BU894/1-3).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Biochemie, Universität zu Köln, Zülpicher Strasse 47, D-50674 Köln, Germany. Phone: 49-221-470-6472. Fax: 49-221-470-5091. E-mail: a.burkovski{at}uni-koeln.de. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, May 2005, p. 2391-2402, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2391-2402.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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