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Journal of Biotechnology 126 (2006) 440–453

Mutation-induced metabolite pool alterations inCorynebacterium glutamicum: Towards the

identification of nitrogen control signals

Tim Muller a,b, Julia Strosser a, Sebastian Buchinger a, Lars Nolden a,1,Astrid Wirtz a, Reinhard Kramer a, Andreas Burkovski a,c,∗

a Institut fur Biochemie, Universitat zu Koln, Germanyb International Max Planck Research School, Max-Planck-Institut fur Zuchtungsforschung, Koln, Germany

c Lehrstuhl fur Mikrobiologie, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Germany

Received 13 February 2006; received in revised form 17 May 2006; accepted 26 May 2006

bstract

The influence of glutamate dehydrogenase activity on nitrogen regulation in Corynebacterium glutamicum was investigated.s shown by RNA hybridization experiments deletion of the gdh gene results in a rearrangement of nitrogen metabolism. Evenhen sufficiently supplied with nitrogen sources, a gdh deletion strain showed the typical nitrogen starvation response of C.
lutamicum. These changes in transcription correlate with distinct alterations of intracellular metabolite pattern. Metabolitenalyses of different mutant strains and the wild type indicated that ammonium and 2-oxoglutarate might influence the nitrogenegulation system of C. glutamicum cells.
2006 Elsevier B.V. All rights reserved.

ontrol;
eywords: Corynebacterium; Glutamate dehydrogenase; Nitrogen c
∗ Corresponding author at: Lehrstuhl fur Mikrobiologie, Friedrich-lexander-Universitat Erlangen-Nurnberg, Staudtstr. 5, 91058rlangen, Germany. Tel.: +49 9131 85 28086;

ax: +49 9131 85 28082.E-mail address: [email protected]

A. Burkovski).1 Present address: Universitat Bonn, Institut fur Rekonstruktiveeurobiologie, Bonn, Germany.

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scigst1

168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2006.05.015

Nitrogen regulation

. Introduction

Corynebacterium glutamicum is a gram-positiveoil bacterium belonging to the group of mycolic acid-ontaining actinomycetes (Chun et al., 1996). It wassolated by Kinoshita et al. (1957) in a screening pro-
ram for l-glutamate-producing bacteria from a soilample collected at Ueno Zoo in Tokyo and at thatime designated as Micrococcus glutamicus (Udaka,960). Today, large amounts of l-glutamate (more than
mailto:[email protected]

dx.doi.org/10.1016/j.jbiotec.2006.05.015

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,500,000 tonnes per year) and l-lysine (more than60,000 tonnes per year) are produced by use of dif-erent C. glutamicum strains, in addition to smallermounts of l-alanine, l-isoleucine and l-proline andn addition to different nucleotides (Leuchtenberger,996; Hermann, 2003).

Due to its pivotal role in glutamate production,asic biochemical work on the glutamate-producingnzymes of C. glutamicum was already started in the960s (Kimura, 1962; Oshima et al., 1964; Shiio andzaki, 1970; Tochikura et al., 1984). As most bacteria,. glutamicum has two primary pathways to facilitate

he incorporation of ammonium into the key nitrogenonors for biosynthetic reactions, l-glutamate and l-lutamine (Shiio and Ozaki, 1970; Jakoby et al., 1997;esch et al., 1998). Glutamate dehydrogenase (GDH)ssimilates ammonium by the reductive amination of 2-xoglutarate to l-glutamate. Alternatively, ammoniums assimilated via the glutamine synthetase/glutamateynthase (GS/GOGAT) pathway, whereby ammoniums used to amidate l-glutamate under consumptionf ATP to form l-glutamine by glutamine synthetaseGS). The amide group is then transferred reductivelyo 2-oxoglutarate by glutamate synthase (GOGAT),esulting in the net conversion of 2-oxoglutarate to l-lutamate. Assimilation via glutamate dehydrogenases bioenergetically more favourable, as the GS/GOGATathway utilizes an additional mol ATP per mol ammo-ium assimilated. Consequently, GDH is preferentiallysed in ammonium-rich medium, while transcriptionf gltBD, coding for GOGAT, is completely repressedn this situation (Beckers et al., 2001; Schulz et al.,002) and transcription of glnA, coding for GS, is main-ained at a basal level to provide l-glutamine for growthNolden et al., 2001a). When the cells face nitrogenimitation, assimilation via GDH is not sufficient due tohe low affinity of the enzyme. In this situation, expres-ion of glnA and gltBD is activated and the GS/GOGATathway is preferentially used for ammonium assimi-ation.

In C. glutamicum the TetR-family transcriptionalegulator AmtR was identified as the master regulatorf nitrogen control (Jakoby et al., 2000; Beckers et al.,005). Under conditions of high nitrogen supply AmtR
locks the transcription of nitrogen-controlled genes,.g. glnA and gltBD. In response to nitrogen limitation,epression is relieved by an interaction of AmtR withhe GlnK signal transduction protein (Beckers et al.,
ucmn

ology 126 (2006) 440–453 441

005). Adenylylation of GlnK by the GlnD protein isrucial for derepression of transcription, since unmod-fied GlnK does not interact with AmtR (Beckers etl., 2005). In response to an improved nitrogen supply,lnD deadenylylates GlnK again, resulting in repres-

ion of AmtR-controlled genes (Strosser et al., 2004).t is unknown how this regulation process is triggeredn C. glutamicum, i.e. which metabolite(s) indicate(s)he nitrogen status of the cell and how this signal isransferred to the GlnD/GlnK/AmtR signal cascade.

In this study, we investigated the influence of glu-amate dehydrogenase activity on nitrogen regulationn C. glutamicum. The gdh gene, encoding glutamateehydrogenase, was isolated by Bormann et al. (1992)nd mutant strains were characterized. Despite the highnzymatic activity of 1.3 U (mg protein)−1 determinedor C. glutamicum wild type GDH (Tesch et al., 1998),eletion of the gdh gene revealed astonishingly noetrimental effect on growth. Moreover, glutamate pro-uction was unaffected as well (Bormann et al., 1992;ormann-El Kholy et al., 1993). Later, Tesch et al.

1998) observed a significantly enhanced activity ofS and GOGAT in the gdh deletion strain under nitro-en surplus, which might complement the lack of GDHctivity. In this communication, we show that the acti-ation of the GS/GOGAT pathway on expression leveln a gdh deletion strain is triggered by characteris-ic changes in the metabolite pool of C. glutamicumeading to a nitrogen starvation response of the globalitrogen regulation network.

. Materials and methods

.1. Bacterial strains and growth conditions

Strains and plasmids used in this study are listedn Table 1. Bacteria were routinely grown in Luriaertani (LB) medium (Sambrook et al., 1989) at0 ◦C (C. glutamicum) or 37 ◦C (Escherichia coli). LBedium for C. glutamicum strains was supplementedith 2% glucose (final concentration) and antibioticsere added in standard concentrations if appropriate

Ausubel et al., 1987). For cell culture, a standard inoc-
lation scheme was applied. A fresh C. glutamicumulture in LB medium was used to inoculate mini-al medium (Keilhauer et al., 1993) with an excess of
itrogen sources (approximately 500 mM ammonium)

442 T. Muller et al. / Journal of Biotechnology 126 (2006) 440–453

Table 1Strains and plasmids used in this study

Strains/plasmids Relevant genotype/description Reference

C. glutamicumATCC 13032 Wild type Abe et al. (1967)LN�GDH ATCC 13032 �gdh This studyLN�GS ATCC 13032 �glnA This study

E. coliDH5�mcr supE44 hsdR17 recA1 endA1 gyrA96 thi1 relA mcrA �(mrr-hsdRMS-mcrBC) Grant et al. (1990)

PlasmidspGEM3z E. coli plasmid for in vitro transcription, ApR Promega, MannheimpGEM4z E. coli plasmid for in vitro transcription, ApR Promega, MannheimpGEMamtA 0.5 kb internal amtA fragment in pGEM3z Jakoby et al. (2000)pGEMamtB 1.0 kb internal amtB fragment in pGEM3z Jakoby et al. (2000)pGEMgdh 0.6 kb internal gdh fragment in pGEM4z This studypGEMglnD 0.7 kb internal glnD fragment in pGEM3z Jakoby et al. (2000)pGEMglnK 0.3 kb internal glnK fragment in pGEM3z Jakoby et al. (2000)pGEMglnA 0.5 kb internal glnA fragment in pGEM3z Nolden et al. (2001b)pGEMgltB 0.5 kb internal gltB fragment in pGEM3z Beckers et al. (2001)pK18mobsacB Vector for generation of unmarked gene deletions, sacB, ApR Schafer et al. (1994)pK18gdh Plasmid pK18mobsacB carrying the flanking DNA regions of the gdh gene This study

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bbreviations: ApR, resistance to ampicillin; KmR, resistance to kan

or overnight growth. This culture, with an overnightD600 of approximately 25–30, was used to inocu-

ate fresh minimal medium to an OD600 of approxi-ately 1.0 and cells were grown until the exponential

rowth phase was reached (OD600 approximately 4–5).o induce nitrogen starvation, cells were harvested byentrifugation and the pellet was suspended in pre-armed minimal medium without nitrogen source. Theitrogen-deprived cells were incubated at 30 ◦C undereration. Subsequently, 200 mM of ammonium (finaloncentration) were added to a culture of nitrogen-tarved cells in order to improve the nitrogen supplygain.

.2. General molecular biology techniques

For plasmid isolation, transformation and cloningteps, standard techniques were used (Ausubel et al.,987; Sambrook et al., 1989). E. coli strain DH5�mcras used as a cloning host. Competent C. glutam-

cum cells were prepared according to van der Restt al. (1999). Chromosomal C. glutamicum DNA was
solated as described (Eikmanns et al., 1994). DNAequence analyses were carried out by the bioanalyt-cs service unit at the Center for Molecular Medicineologne.
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DNA regions of the glnA gene This study

.

.3. Construction of RNA probes, RNAreparation and northern hybridization analysis

An internal 0.6 kb gdh fragment was amplified viaCR using chromosomal DNA from ATCC 13032s template. For subsequent cloning steps, XbaInd EcoRI restriction sites (shown in bold) werentroduced in the primer sequences (5′-GCGCGCCTAGAGTGCGAGCCTGAGCGTCA-3′/5′-GCG-GCGAATTCGGAGCTTAGCCACGTCAA-3′).fter restriction of the PCR product with EcoRI andbaI, the gdh fragment was ligated to vector pGEM4z

Promega, Heidelberg), leading to plasmid pGEMgdh.fter linearization by XbaI this DNA was used for in

itro transcription to generate a dioxigenin-labelledNA probe using SP6 RNA polymerase and the DIGNA Labelling Mix (Roche Diagnostics, Mannheim).

Total RNA was prepared after disruption of the. glutamicum cells by glass beads using the Nucleopin RNA II Kit as recommended by the sup-lier (Macherey-Nagel, Duren). The RNA was spot-ed directly onto nylon membranes using a Schle-
cher and Schuell (Dassel) Minifold I Dot Blotter.ybridization of digoxigenin-labelled RNA probesas detected with a Fuji luminescent image anal-ser LAS1000 or Kodak X-OMAT X-ray films using

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lkaline phosphatise-conjugated anti-digoxigenin fabragments and CSPD as light-emitting substrate asecommended by the supplier (Roche Diagnostics,

annheim).

.4. Construction of gdh and glnA deletion strains

Unmarked chromosomal deletions were introducednto the C. glutamicum genome using the protocolescribed by Schafer et al. (1994). In order to generategdh deletion, a DNA fragment spanning the gdh

ene together with its flanking sequences (1.5 kb up-nd downstream of gdh) was amplified via PCR usinghromosomal DNA from ATCC 13032 as template.or subsequent cloning steps, HindIII and EcoRIestriction sites (shown in bold) were introduced in therimer sequences (5′-GCGCGCGAATTCATCAA-ATCGTCAGCAGTGG-3′/5′-GCGCGCAAGCTT-TGTGGATCGGATAATTGACCTG-3′). After res-

riction with EcoRI and HindIII, the PCR productas ligated to vector pK18mobsacB. A 500 bpcoRV/BamHI fragment of the gdh gene was removed

rom the plasmid. After treatment with Mung Beanuclease (NEB, Schwalbach), the plasmid was re-

igated. The resulting plasmid, pK18�gdh, was usedor the chromosomal deletion via two independentecombination events as described (Schafer et al.,994).

For construction of a chromosomal glnA deletion, aNA fragment spanning the glnA gene together with

ts flanking sequences (2.8 kb) was amplified via PCRsing chromosomal DNA from ATCC 13032 as tem-late. For subsequent cloning steps, XbaI and HindIIIestriction sites (shown in bold) were introduced in therimer sequences (5′-GCGCGCTCTAGAATCAAG-TCGTCAGCAGTGG-3′/5′-GCGCGCAAGCTTT-GTTAACCTCAAAGGCATCGACG-3′). After rest-

iction with XbaI and HindIII, the PCR productas ligated to vector pUC19. A 1.2 kb BsmI/BglII

ragment of the glnA gene was removed from thelasmid. After treatment with Mung Bean NucleaseNEB, Schwalbach), the plasmid was re-ligated.he resulting plasmid was restricted with XbaI andindIII and the 1.6 kb insert was ligated to vector
K18mobsacB. The resulting plasmid, pK18�glnA,as used for the chromosomal deletion via two inde-endent recombination events as described (Schafer etl., 1994).
C0nl

ology 126 (2006) 440–453 443

.5. Western blotting

For the preparation of C. glutamicum cell extracts,liquots were transferred from the culture to tubesontaining glass beads and immediately frozen in liq-id nitrogen. Samples thawed on ice were disruptedsing a FastPrep FP120 instrument (Q-BIOgene, Hei-elberg) placed in a cooling chamber. Subsequently,lass beads and cell debris were removed by centrifu-ation. Protein concentrations were determined usingoti-Nanoquant as recommended by the supplier (Carloth, Karlsruhe) using bovine serum albumin as a

tandard. SDS-PAGE was carried out using 12% acry-amide gels and a tricine buffer system as described bychagger and von Jagow (1987). After electrophore-is, the gel-separated proteins were transferred ontopolyvinylidene difluoride membrane (PVDF; Carloth, Karlsruhe) by electroblotting and incubated withlnK-specific antiserum. Antibody binding was visu-

lized by appropriate secondary antibodies coupled tolkaline phosphatase or peroxidase (Sigma–Aldrich,armstadt) and the BCIP/NBT alkaline phosphatase

ubstrate (Sigma–Aldrich, Darmstadt).

.6. Determination of internal 2-oxoglutarate and-glutamate

Ten milliliters of a C. glutamicum culture (OD600pproximately 4–5) were harvested by centrifuga-ion (10 min, 4000 × g). The supernatant was removednd the cell pellet was washed with pre-warmedresh culture medium and then re-suspended in 1 mlethanol. After incubation for 20 min at 70 ◦C, cell

ebris was removed by centrifugation. The super-atant was dried at 60 ◦C under continuous nitro-en flow. The residue was dissolved in 50 �l ofsolution of 20 g/l methoxylamine in pyridine and

ncubated for 90 min at 30 ◦C. Eighty microlitersf N-methyltrimethylsilyltrifluoroacetamide (MSTFA)ere added followed by an incubation of 60 min at5 ◦C. The concentration of derivatized 2-oxoglutaratend l-glutamate were measured by gas chromatog-aphy using the TraceGC system (Thermo Finni-an, Woburn) and a FS-Supreme 5 column (CS-
hromatographie, Langenwehe). After an injection of.3 �l (split ratio 1:25), separation was achieved underitrogen gas flow (flow rate: 1 ml/min) using the fol-owing time program: 2 min at 60 ◦C, temperature gra-

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44 T. Muller et al. / Journal of B

ient of 30 ◦C/min to 140 ◦C, temperature gradient of◦C/min to 175 ◦C, temperature gradient of 30 ◦C/min

o 320 ◦C, 5 min at 320 ◦C. At the end of the run, initialonditions were restored. Column effluents were mon-tored by flame ionization detection (FID) at 300 ◦C.nternal concentration were calculated from the datasing a dry weight of 0.35 mg/ml OD600 = 1 and annternal volume of 2.0 �l/mg dry weight.

.7. Determination of ammonium

Two microliters of a C. glutamicum culture (OD600pproximately 4–5) were harvested by centrifugation5 min, 13,000 × g). The weight of the pellet was deter-ined and the cells were resuspended in 100 �l of

n aqueous 0.1% N-cetyl-N,N,N-trimethylammoniumromide (CTAB) solution. After incubation for 10 mint room temperature, cell debris was removed byentrifugation. The ammonium concentrations of thisupernatant (cell extract) as well as of the super-atant obtained in the first centrifugation step (cul-ure medium) were determined by reversed-phasePLC using a protocol based on the method describedy Lindroth and Mopper (1979). Ammonium wasabelled by pre-column derivatisation using ortho-hthaldialdehyde (OPA). For separation, the Agilent100 system (Agilent, Waldbronn) with a LiChrospherP 18 (150 mm × 3 mm, 5 mm) column (Merck, Darm-

tadt) and 60% methanol as mobile phase with a lowate of 0.8 ml/min at 40 ◦C were used. Column effluentsere monitored with a G1321A Fluorimenter (Agilent,aldbronn) at an excitation wavelength of 330 nm anddetection wavelength of 455 nm.

.8. Calculation of intracellular ammoniumoncentrations

From the ammonium concentrations of cell extractnd culture medium measured by HPLC, the intracel-ular ammonium concentration can be calculated. As arerequisite for this calculation, the ratio of internal cellolume to total pellet volume had to be determined. Forhis purpose, the distribution of a membrane permeablerobe, [3H]-labelled water, a membrane impermeable
ne, and [14C]-labelled taurine, was measured. About2.0 ± 1.0% of the cell pellet consisted of surroundingedium. Internal concentration were calculated from
he data using a dry weight of 0.35 mg/ml OD600 = 1

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ology 126 (2006) 440–453

nd an internal volume of 2.0 �l/mg dry weight (foretermination of cytoplasmic volumes depending onedia composition, see Ronsch et al., 2003).

.9. Determination of internal l-glutamine

One milliliter of a C. glutamicum cell culture (OD600pproximately 4–5) was separated from the surround-ng medium by rapid filtration (within less than 30 s).he cells were washed with pre-warmed fresh cul-

ure medium and then re-suspended in 1 ml of anqueous solution of 0.1% CTAB. After incubation for0 min at room temperature, cell debris was removedy centrifugation. The l-glutamine concentration of theupernatant was determined by reversed-phase HPLC.-Glutamine was labelled by pre-column derivatisa-ion using ortho-phthaldialdehyde (OPA). For sep-ration, the Agilent 1100 system (Agilent, Wald-ronn) with a 125/4 Nucleodur 100-5 C18 ec columnMacherey-Nagel, Duren) was used. The followinguffer system was applied: (A) 40 mM sodium acetateuffer (pH 6.5):methanol:acetonitrile (95:2.5:2.5); (B)ethanol:acteonitrile (50:50). Column effluents wereonitored with a G1321A fluorimeter (Agilent, Wald-

ronn) at an excitation wavelength of 330 nm and aetection wavelength of 455 nm. Internal concentra-ion were calculated from the data using a dry weightf 0.35 mg/ml OD600 = 1 and an internal volume of.0 �l/mg dry weight.

.10. Metabolome analysis by gashromatography/mass spectrometry

Sample preparation and mass spectrometry was car-ied out using the protocol described by Strelkov etl. (2004) with slight modifications. In short, approxi-ately 5 × 1010 cells were harvested at the exponential

rowth phase. Samples were centrifuged (4500 × g,min, 4 ◦C) and cells were washed with 20 ml 0.9%aCl at 4 ◦C. Intracellular metabolites were extractedy ultrasonification of the harvested cells for 15 min at0 ◦C in 1.5 ml methanol (containing 8 mg/ml ribitols internal standard). Samples were chilled on iceor 2 min, 1.5 ml water was added and the samples
ere mixed thoroughly. Subsequently, 1 ml of chlo-
oform was added and samples were mixed again inrder to remove lipophilic compounds from the polarhase. Finally, phases were separated by centrifugation

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min at 3900 × g and 1 ml of the polar methanol/waterhase was transferred into a new 2 ml sample tube andried under vacuum (Speedvac Concentrator, Eppen-orf, Hamburg, Germany).

For derivatization dried samples were resolved in0 �l pyridine (Fluka–Sigma–Aldrich Co., Taufkir-hen, Germany) containing 20 mg/ml methoxyamineFluka–Sigma–Aldrich Co., Taufkirchen, Germany)nd incubated for 90 min at 30 ◦C under agitation.ubsequently, 32 �l N-methyl-N-trimethylsilyltri-uoroacetamide (MSTFA, CS Chromatographyervice GmbH, Langerwehe, Germany) were added,ollowed by an incubation for 30 min at 37 ◦C and 2 h at5 ◦C. Four microliters of a mixture of eight differentlkanes (C10, C12, C15, C19, C22, C28, C32, C36, 0.2%n cyclohexane each) was added for the calibration ofetention indices according to van den Dool and Kratz1963). Two microliters of the derivatized samplesere injected in a Finnigan Trace Gas Chromatograph

ThermoFinnigan, San Jose, USA) equipped with aB-5MS column (J&W Scientific, Folsom, USA).luted compounds were analysed with a Trace Masspectrometer (ThermoFinnigan, San Jose, USA) afterlectron impact ionization. All parameters for samplenjection, gas chromatography and mass spectrometryere described elsewhere (Strelkov et al., 2004).

. Results

.1. Deletion of the gdh gene affects nitrogenontrol

Tesch et al. (1998) showed previously high GOGATctivity in a glutamate dehydrogenase-deficient strainithout studying the molecular background of thisbservation. We assumed that glutamate synthase acti-ation occurs on the level of transcription and, there-ore, tested transcription of the gltB gene. For thisurpose, RNA was isolated from the wild type ATCC3032 and gdh deletion strain LN�GDH, grownnder conditions of high nitrogen supply (approxi-ately 500 mM ammonium), during nitrogen starva-

ion and after addition of ammonium to nitrogen-
tarved cells. The RNA hybridization experiments car-ied out revealed that deletion of gdh leads to the dereg-lation of transcription of gltB (Fig. 1A). This resultxplains the observed phenotype of a gdh mutant strain
mtis

ology 126 (2006) 440–453 445

nd the high glutamate synthase activity observed byesch et al. (1998).

In order to elucidate whether gltB deregulation isaused by a specific effect or by a so far unknownerepression of the AmtR regulon in spite of nitro-en surplus conditions, additionally, expression ofther AmtR-controlled genes was investigated. In fact,ranscription of glnA, encoding glutamine synthetase,lnD, coding for the GlnK-modifying/demodifyingnzyme, glnK, encoding the central nitrogen signalransduction protein GlnK, and the ammonium trans-orter encoding amtA and amtB genes were tested andound to be derepressed in the gdh deletion strain (forltB, glnA and glnD expression see Fig. 1A, data forther genes not shown).

For the release of the AmtR repressor from its targetNA, an interaction with adenylylated GlnK protein is

rucial (Beckers et al., 2005). Typically, GlnK is syn-hesized and adenylylated during nitrogen starvationnly (Nolden et al., 2001b; Strosser et al., 2004). To testhether the gdh deletion influences GlnK synthesis andodification, Western blot analyses were carried out.hese experiments showed that GlnK was in contrast to

he wild type present in high amounts and at least is par-ially adenylylated in the gdh mutant LN�GDH evennder nitrogen surplus conditions (Fig. 1B), explaininghe transcription of AmtR-regulated genes.

The reason for the induction of nitrogen starvationesponse in a gdh deletion strain was unclear. Since wessumed that the deregulatory effect of a gdh deletionight be caused by changes in the concentration of aetabolite, which serves as an indicator for the nitrogen

tatus of the cell, we investigated the concentrations ofifferent metabolites in wild type and mutant strain.

.2. Influence of glutamate dehydrogenase onntracellular metabolite pools

Glutamate dehydrogenase activity influences theoncentrations of its product glutamate and its educts-oxoglutarate and ammonium. Indirectly, also the l-lutamine concentration might be influenced. Con-equently, we focused on the determination of theseetabolites in order to elucidate which metabolite
ight be responsible for deregulation of nitrogen con-
rol in C. glutamicum. For this purpose C. glutam-cum wild type strain ATCC 13032 and gdh deletiontrain LN�GDH were cultivated in nitrogen-rich min-


Fig. 1. (A) RNA dot blot hybridization experiment. Total RNA was isolated from wild type ATCC 13032 and gdh deletion strain LN�GDHcultivated under nitrogen excess (+N), after 15, respectively, 30 min of nitrogen starvation (−N15, −N30) and 10 min after an excess ofammonium was added (N pulse 10). The RNA was hybridized with antisense probes directed against gltB, glnA and glnD transcripts. (B) GlnKsynthesis and modification in strain ATCC 13032 and LN�GDH. Cytoplasmic proteins were prepared from cells grown in nitrogen-rich medium(1) and nitrogen-deprived cells (2), separated by SDS-PAGE (25 �g/lane), blotted and probed with antibodies directed against nitrogen signalt lylated( ker bro

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ransduction protein GlnK. The upper band corresponds to the adeny3) Molecular weight marker (MBI fermentas prestained proteinmar

mal medium and the internal concentrations of 2-xoglutarate, ammonium, glutamate and glutamineere measured by HPLC and gas chromatography,

espectively.In the two strains, almost identical l-glutamine con-

entrations (about 15 mM) were observed (Fig. 2A).-Glutamate and ammonium concentrations were dif-ering between ATCC 13032 and LN�GDH (Fig. 2Bnd C). However, these concentrations were very high
n any case, making them a unlikely signal for a starva-ion condition. Only the 2-oxoglutarate concentrationn strain LN�GDH exceeded the wild type concentra-ion by a factor of 3.5 (Fig. 2D) making 2-oxoglutarate
sttm

protein, the lower band to unmodified GlnK (Strosser et al., 2004).ad range, from top to bottom: 70, 55, 45, 35, 25, 15 and 10 kDa).

likely candidate for the deregulation of nitrogen con-rol in the gdh deletion strain.

To test, if 2-oxoglutarate is also a marker of nitro-en supply in the wild type, ATCC 13032 cells weretarved for nitrogen sources and internal solute concen-rations were determined. In the wild type, starvationor 30 min resulted only in a slight decrease of the glu-amate pool from 143 ± 18 to 98 ± 16 mM (Fig. 3B).his observation is in accord with previous data, which
howed that the glutamate pool reacts only very slowlyo nitrogen starvation (Nolden et al., 2001b). Sincehe transcriptional response to starvation occurs within
inutes, glutamate can be excluded as an indicator

T. Muller et al. / Journal of Biotechnology 126 (2006) 440–453 447

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ig. 2. Intracellular metabolite concentrations in ATCC 13032 andlutamate (B), 2-oxoglutarate (C) and ammonium (D) were determineells grown in nitrogen-rich minimal medium.

f the nitrogen status of the cell. Interestingly, alsohe internal concentration of 2-oxoglutarate was con-tantly at 1.2 mM under nitrogen surplus as well asnder nitrogen starvation in the wild type (Fig. 3C).n contrast, the internal concentrations of l-glutaminend ammonium were high during nitrogen surplus butignificantly diminished in response to nitrogen star-ation. Glutamine concentrations decreased from 14.4o 1.7 mM, while ammonium concentrations decreasedrom 152 to 2 mM (Fig. 3A and D). Thus, the internaloncentrations of ammonium and glutamine correlatedith the nitrogen supply, i.e. the availability of ammo-ium in the surrounding medium, while no changesf 2-oxoglutarate were observed in the C. glutamicum
ild type under the tested conditions. This discrep-
ncy between gdh deletion and wild type strain mighte explained by their different ammonium assimila-ion capacity. While in the gdh deletion strain nitrogen

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ig. 3. Intracellular metabolite concentrations in strain ATCC 13032 depe-glutamine (A), l-glutamate (B), 2-oxoglutarate (C) and ammonium (D)ild type cells grown under nitrogen surplus (+N) and incubated in nitrogen

DH cells. The intracellular concentrations of l-glutamine (A), l-s chromatography and HPLC analysis from wild type and LN�GDH

etabolism is at least partially blocked, this is not thease in the wild type. Therefore, sampling times usedor experiment might be too slow to detect regulatorymportant transient changes of metabolites in the wildype.

.3. Analysis of metabolites and GlnKdenylylation state

For more time-resolved analysis of the wild typeitrogen starvation response, cells were grown initrogen-rich minimal medium, transferred to nitrogen-ree medium and then treated with an ammonium pulse.t different time points samples were analyzed in

espect to metabolite alterations, gltB expression andlnK adenylylation state.To determine metabolite concentrations, gas chro-

atography/mass spectrometry was used. This method

nding on the nitrogen supply. The intracellular concentrations ofwere determined by gas chromatography and HPLC analysis from-free minimal medium for 30 min (−N).

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s well suited to detect relative changes caused by muta-ions or changed environmental conditions (Strelkov etl., 2004). When cells were harvested and re-suspendedn nitrogen-free minimal medium, a slight increase of-oxoglutarate was observed, most likely due to theentrifugation step. During the next 30 min of nitrogentarvation, the 2-oxoglutarate pool increased steadilyp to a factor of about 30. In response to the additionf ammonium, the initial level of 2-oxoglutarate waseached almost immediately. Adenylylation of GlnKnd transcription of gltB were correlated to a low 2-xoglutarate pool. As a consequence, it is very likelyhat 2-oxoglutarate indicates the nitrogen supply of theild type as well. A similar correlation of l-glutamate

nd l-glutamine was not found. However, due to thexperimental design, GlnK adenylylation was also cor-elated with ammonium availability. It is noteworthy inhis respect that nitrogen starvation response is not fullyctivated in strain LN�GDH grown in ammonium-richedium (Fig. 1B). Under these conditions a consider-

ble amount of unmodified GlnK was observed in theells. Therefore, a contribution to nitrogen regulationf both, 2-oxoglutarate and ammonium can be assumedFig. 4).

.4. A marker for nitrogen supply: l-glutamineersus ammonium

In enterobacteria, 2-oxoglutarate is a marker ofitrogen supply besides l-glutamine. As indicated byhe data presented here and by previously publishedxperiments (Nolden et al., 2001b) in C. glutam-cum ammonium rather than l-glutamine might be

second effector molecule which indicates the cel-ular nitrogen status. To support this hypothesis, wegain used a mutant strain with blocked ammoniumetabolism, in this case glnA deletion strain LN�GS,hich lacks glutamine synthetase and is as a con-

equence glutamine-auxotrophic. LN�GS cells wereultivated in medium containing ammonium and glu-amine as nitrogen sources and subsequently incubatedn medium without any nitrogen source for 30 min.fter this time, ammonium, glutamine or ammonium

nd glutamine together were added to the nitrogen-
tarved cells, which were then cultivated for another0 min. Transcription of the gltB gene was analyzed byNA dot blot hybridization (Fig. 5A). In addition, the
nternal concentrations of l-glutamine, ammonium, 2-

tb2i

ology 126 (2006) 440–453

xoglutarate and l-glutamate were measured by HPLCnd GC, respectively.

The presence of ammonium correlated with theranscription level of gltB under all tested conditionsFig. 5A and B). Transcription of gltB was repressednder high internal concentrations of ammonium+gln + NH4

+: 604 mM; gln−NH4+-pulse: 478 mM;

H4+-pulse: 450 mM), and activated under low inter-

al concentrations of ammonium (−N: 24 mM; gln-ulse: 26 mM). In contrast, the internal concentrationf glutamine was not correlated to the transcriptionevel of gltB, thus, was not decisive for nitrogen con-rol (Fig. 5A and C). After the addition of ammo-ium to a culture of nitrogen-starved cells, the glu-amine pool was almost unaltered compared to nitro-en starvation (−N: 6.8 mM glutamine; NH4

+-pulse:0.3 mM glutamine), nevertheless, transcription of gltBas repressed. Vice versa, gltB was still transcribed

fter the addition of glutamine to a culture of nitrogen-tarved cells even though the internal concentration oflutamine increased significantly to 81 mM. In the glnAeletion strain the internal 2-oxoglutarate concentra-ion was low (1.05 mM) 30 min after the glutamineulse (Fig. 5D), which might be explained by thection of glutamate synthase converting glutamine and-oxoglutarate to glutamate. As indicated by the inves-igation of strain LN�GDH (Fig. 1B), 2-oxoglutarateeems not function as the only effector molecule in C.lutamicum. Glutamate could again be excluded as arigger of nitrogen control since its internal concen-ration was more or less unaltered under the testedonditions (Fig. 5E).

.5. External versus internal ammonium as autative indicator of nitrogen supply

Due to the experimental design and the membraneermeability of ammonium in its uncharged formmmonia, it was unclear if the internal or the externaloncentration of ammonium was sensed by C. glu-amicum, as both correlated with the transcription ofltB. This question was addressed by a combinationf RNA hybridization and feeding experiments. Whilemmonium is the preferred nitrogen source of C. glu-
amicum, also other nitrogen sources are metabolizedy this organism (for review, see Burkovski, 2003a,b,005). These can be used to test whether external ornternal ammonium indicates nitrogen supply in a C.

T. Muller et al. / Journal of Biotechnology 126 (2006) 440–453 449

Fig. 4. Transient alterations of GlnK modification, gltB transcription and metabolite pools. Wild type strain ATCC 13032 was cultivated innitrogen-rich minimal medium, harvested and transferred to nitrogen-free minimal medium and subsequently treated with an ammonium pulse(200 mM). Synthesis and adenylylation of GlnK was monitored by Western blotting, gltB transcription by RNA hybridization experiments andmetabolite pools were analyzed by gas chromatography/mass spectrometry. Samples were taken from nitrogen-supplied cells (1), immediately,5, 15 and 30 min after transfer in nitrogen-free medium (2–5; for medium exchange cells were harvested by 5 min of centrifugation) andimmediately, 5 and 15 min after addition of 200 mM ammonium (6–8).


Fig. 5. Transcription of gdh and intracellular metabolite concentrations in glnA strain LN�GS. The glnA deletion strain LN�GS was cultivatedin medium containing ammonium and glutamine as nitrogen sources (+gln + NH4

+). Then, the cells were cultivated in medium without anynitrogen source for 30 min (−N). After that, either ammonium (NH4

+-pulse) or glutamine (gln-pulse) or ammonium and glutamine (gln−NH4+-

pulse) were added to the nitrogen-starved cells, which were then cultivated for another 30 min. Total RNA was isolated and analyzed by RNAdot blot hybridization using gltB as probe (A). The internal concentrations of ammonium (B), glutamine (C), 2-oxoglutarate (D), and glutamate(E) as well as the external concentration of ammonium (F) were measured by HPLC and GC, respectively.

T. Muller et al. / Journal of Biotechn

Fig. 6. Influence of different nitrogen sources on the expression ofAmtR-regulierted genes. Total RNA was isolated from wild typeATCC 13032 after incubation in nitrogen-free medium for 1.5 h (1)ac2

gnTbccddf(sssatt

4

iot2galtlpo1w

sAapgao

awaulOlsdiangscs

nmir2ftbHHsAwAp

A

nd after addition of 100 mM ammonium sulfate (A), urea (B) andreatinine (C), respectively, and incubation for 5, 10, 20, 30 min, 1,and 4 h (2–8). The RNA was hybridized with a gltB probe.

lutamicum cell. For this purpose, urea and creati-ine were chosen and added to nitrogen-starved cells.hese two nutrients are not extracellularly degradedut are transported into the cell and decomposed intra-ellularly either by urease leading to ammonium andarbon dioxide (Siewe et al., 1998) or by creatinineeaminase (Bendt et al., 2004) leading to methylhy-antoin and ammonium inside the cell. As a markeror nitrogen limitation, gltB expression was monitoredFig. 6). RNA hybridization revealed that gltB is tran-cribed in the wild type in the absence of nitrogenources, while ammonium addition blocks gltB expres-ion within 10 min. Interestingly, also addition of ureand creatinine blocks transcription, which indicateshat internal rather than external ammonium indicateshe nitrogen supply.

. Discussion

When C. glutamicum wild type cells are growingn nitrogen-rich standard minimal media, transcriptionf gltBD and glnA is repressed by the master regula-or of nitrogen control, AmtR (Beckers et al., 2001,005; Nolden et al., 2001a). In a gdh mutant strainrown under high ammonium supply this would lead tosituation in which ammonium assimilation would be

imited to low glutamine synthetase activity while glu-amate generation would be highly impaired due to theack of gltBD transcription. However, it was reported
reviously that deletion of gdh has no obvious effectn growth and glutamate production (Bormann et al.,992; Bormann-El Kholy et al., 1993). In this study,e found that a C. glutamicum gdh deletion strain
ldS

ology 126 (2006) 440–453 451

hows transcription of genes typically repressed bymtR during nitrogen surplus, including the glnA gene

nd the gltBD operon which encode the GS/GOGATathway for ammonium assimilation. Thus, deletion ofdh causes relief of AmtR-dependent repression, whichllows circumventing the putatively detrimental effectsf a lacking glutamate dehydrogenase.

Central metabolites of nitrogen metabolism, i.e.mmonium, glutamine, glutamate and 2-oxoglutarate,ere analyzed in the wild type and in strains LN�GDH

nd LN�GS. Based on these investigations, it is verynlikely that glutamate and glutamine indicate the cel-ular nitrogen status in C. glutamicum wild type cells.ur studies hint to 2-oxoglutarate as marker metabo-

ite that indicates nitrogen supply of C. glutamicum,ince the release of repression by AmtR in the gdheletion strain under nitrogen surplus was correlated tontracellular accumulation of 2-oxoglutarate due to thebsence of 2-oxoglutarate-consuming GDH. Ammo-ium seems to be a second effector molecule of nitro-en regulation. Data obtained by the feeding of nitrogenources, which are intracellularly decomposed, indi-ate that internal rather than external ammonium isensed by C. glutamicum.

The sensory proteins responsible to monitor theitrogen status in C. glutamicum are still unknown. Theost likely candidate for the sensing of 2-oxoglutarate

s the PII-type signal transduction protein GlnK (foreview, see Arcondeguy et al., 2001; Ninfa and Jiang,005). Future binding assays with the isolated proteinrom C. glutamicum might show whether this pro-ein in fact binds 2-oxoglutarate as shown in otheracteria. For Saccharomyces cerevisiae (Lorenz andeitman, 1998), Rhodobacter capsulatus (Yakunin andallenbeck, 2000) and E. coli (Javelle et al., 2004) a

ensory function of Mep/Amt proteins was discussed.lso in this case, new efforts are necessary to show,hether C. glutamicum Amt proteins AmtA and/ormtB are involved in ammonium sensing or if an inde-endent sensor mechanism is realized in this bacterium.

cknowledgements

The authors wish to thank Anja Wittmann for excel-ent technical assistance, George Coupland for helpfuliscussions and interest in the project and Dietmarchomburg for support.

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