Institut für Biochemie der Universität zu Köln, Zülpicher-Str. 47, D-50674 Köln, Germany1
Author for correspondence: Andreas Burkovski. Tel: +49 221 470 6472. Fax: +49 221 470 5091. e-mail: a.burkovski{at}uni-koeln.de
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ABSTRACT |
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Keywords: GOGAT, nitrogen control, nitrogen starvation
Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase
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INTRODUCTION |
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Corynebacterium glutamicum is a Gram-positive soil bacterium, which belongs phylogenetically to the actinomycetes group and is related to important pathogens like Corynebacterium diphtheriae, Mycobacterium leprae and Mycobacterium tuberculosis, and to the antibiotic-producing streptomycetes. Due to its remarkable ability to excrete high amounts of glutamic acid under conditions of biotin limitation (Kinoshita et al., 1957 ; Gutmann et al., 1992
), this bacterium is applied in fermentation processes on an industrial scale. By use of different C. glutamicum mutant strains, not only large amounts of L-glutamate (1000000 t per year) but also L-lysine (450000 t per year) are produced, in addition to smaller amounts of the industrially less important amino acids L-alanine, L-isoleucine and L-proline (Leuchtenberger, 1996
).
Due to their putatively pivotal role in glutamate production, basic biochemical work on the ammonium-assimilating enzymes GDH, GS and GOGAT was carried out earlier (Shiio & Ozaki, 1970 ; Tachiki et al., 1981
; Tochikura et al., 1984
). However, with the isolation of the corresponding genes, a more detailed characterization of the different enzymes and their regulation was possible. The gdh gene was isolated by Börmann et al. (1992)
. Deletion and overexpression of this gene revealed that GDH is not essential for glutamate formation with respect to growth and glutamate production in C. glutamicum (Börmann-El Kholy et al., 1993
). GDH activity is not regulated in dependence on nitrogen availability, i.e. no alterations in specific enzyme activity were found when raising the ammonium concentration in the growth medium from 1 to 90 mM (Tesch et al., 1998
). The glnA gene, encoding GS I, was isolated by Jakoby et al. (1997)
. Site-directed mutagenesis experiments revealed that GS is regulated via adenylylation/deadenylylation. Whilst nitrogen starvation enhances GS activity (Jakoby et al., 1999
; Tesch et al., 1999
), the enzyme is down-regulated by adenylylation upon addition of ammonium (Jakoby et al., 1997
). However, in vivo flux analysis by 15N nuclear magnetic resonance showed that also in the presence of 100 mM ammonium a surprising large fraction, 28%, of ammonium is assimilated via GS. It was assumed that the observed high GS activity is the result of a strong demand for glutamine as a nitrogen donor in amidotransferase reactions for cell-wall synthesis (Tesch et al., 1999
). In contrast to GS, GOGAT is inactive when cells were grown with high ammonium supply (Tesch et al., 1999
). The nucleotide sequence of the genes encoding the large and small GOGAT subunits
and ß, gltB and gltD, was published in GenBank by Kanno and co-workers in 1999 (accession no. AB024708) without further experimental evidence.
In this study we present data regarding the genetic organization of the gltB and gltD genes, their transcriptional regulation, and the effect of inactivation of these genes on GOGAT activity, growth with different nitrogen sources, methylammonium/ammonium uptake and glutamate production.
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METHODS |
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RNA preparation and Northern hybridization analysis.
Total RNA was prepared after disruption of the C. glutamicum cells by glass beads using the RNeasy Mini Kit as recommended by the supplier (Qiagen). The RNA was either size-fractionated using agarose gels containing formaldehyde and blotted onto positively charged nylon membranes (Sambrook et al., 1989 ) or spotted directly onto nylon membranes using a Schleicher & Schuell Minifold I Dot Blotter. Hybridization of digoxigenin-labelled RNA probes was detected with a Fuji luminescent image analyser LAS1000 or Kodak X-OMAT X-ray films using alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments and CSPD as light-emitting substrate as recommended by the supplier (Roche Diagnostics).
Digoxigenin labelling of DNA and gel retardation experiments.
Digoxigenin-labelled DNA fragments were prepared using digoxigenin-labelled primers for PCR (5'-DIG-CAAGTCGGGCTGCGATGG- 3' / 5' -DIG-AATCAGGCGCAGTACCTGC-3'). The amtR gene was heterologously expressed in E. coli DH5mcr pUC11-1.8 and cell extracts were prepared by ultrasonic treatment. For the retardation experiments, digoxigenin-labelled PCR product was incubated in TEK buffer (10 mM Tris/HCl, pH 8·0, 10 mM KCl, 1 mM EDTA, 7 µM 2-mercaptoethanol) containing 200 µg BSA ml-1 together with salmon sperm DNA (150 µg ml-1) and various amounts of cell extract for 15 min on ice. The samples were loaded onto a 4% polyacrylamide gel, and electrophoresis and blotting were subsequently carried out as described by Sambrook et al. (1989)
. Bands were detected with a Fuji luminescent image analyser LAS1000 or Kodak X-OMAT X-ray films using alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments to probe the digoxigenin-labelled DNA and CSPD as light-emitting alkaline phosphatase substrate, as recommended by the supplier (Roche Diagnostics).
Construction of glt mutant strains.
For the disruption of glt genes in the C. glutamicum chromosome, internal gene fragments of gltB and gltD were amplified by PCR using chromosomal DNA of the wild-type as template and the primer combinations 5'-CCGTGGCGCGCAGCTTGC-3'/5'-CCCATGGACCACCGGCATG-3' for gltB and 5'-AGGCGCCTGCGTGCTCGG- 3 ' / 5 ' -GCGGTTTTGGGCGGTGAG-3' for gltD. The resulting 1·0 kb gltB and 0·5 kb gltD fragments were ligated to plasmid pK18mob. After transfer of these constructs in competent C. glutamicum cells via electroporation (van der Rest et al., 1999 ), kanamycin-resistant clones carried the plasmids integrated in the chromosome via homologous recombination since pK18mob derivatives cannot be replicated by C. glutamicum.
Construction of a lacZ reporter gene strain.
As the first step to exchange the chromosomal gltB gene for a gltBlacZ fusion, a DNA fragment comprising 564 bp upstream of the gltB gene and its first 593 bp was amplified via PCR using the following primers: 5'-CCGGAATCGTCTTTCAGGATC-3'/5'-GCATGCAGCAATGGCCGACTCCAGGC-3'. After phosphorylation, the PCR product was ligated to SmaI-restricted and dephosphorylated pUC18 DNA. In the resulting plasmid, pUCgltB, a XbaI and a XhoI restriction site were introduced directly downstream of position +6 via site-directed mutagenesis, leading to plasmid pUCgltBXbaI/XhoI. In the second step, the lacZ gene was amplified by PCR from plasmid pLacZi. With the primers 5'-TCTAGAATGACCGGATCCGGAGCTTG- 3 ' / 5 ' -CATGAGTTACGCGAAATACGGGCAGAC-3', a XbaI site was introduced at the 5' end and a XhoI site at the 3' end of the DNA fragment (shown in bold). The XbaI/XhoI-restricted PCR product was ligated to XbaI/XhoI-restricted and dephosphorylated pUCgltBXbaI/XhoI plasmid DNA, leading to pUCgltBlacZ. In the last step, the gltBlacZ fusion was isolated by EcoRI/SphI restriction and cloned into vector pK18mobsacB. Integration of the resulting plasmid pK18mobsacBgltBlacZ in the C. glutamicum chromosome and allelic exchange was carried out as described by Schäfer et al. (1994) . The correct chromosomal integration of the lacZ fusion in strain GltBlacZ was checked by PCR (data not shown).
Enzyme-activity measurements, transport assays and miscellaneous methods.
Cell extracts were prepared by ultrasonic treatment. The protein content of samples was determined using a modified Bradford assay (Roti-Quant, Roth). GOGAT activity was measured as described by Meers et al. (1970) , ß-galactosidase activity was assayed as described by Miller (1972)
using cells permeabilized with 0·2% (final concentration) N-cetyl-N,N,N-trimethylammonium bromide (CTAB). Glutamate in the culture supernatant was determined according to Gutmann et al. (1992)
. Uptake of [14C]methylammonium was measured using a rapid filtration approach (Siewe et al., 1996
).
Computer-assisted DNA analyses.
Database searches were performed using the BLAST program (Altschul et al., 1990 ). For domain analyses the PROSITE software package (http//:www.expasy.ch) and the Pfam program (http//:www.sanger.ac.uk) were used.
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RESULTS |
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From the deduced amino acid sequence, a number of putative functional important amino acid motifs could be identified (for overview, see Fig. 1). As, for example, in the Azospirillum brasilense GOGAT (Pelanda et al., 1993
), six regions with the sequence GXG2(G/A/P), which interact with the adenylate portion of FAD or NADPH, were observed in the
subunit (gltB gene product) and two in the ß subunit (gltD gene product). Bacterial GOGATs contain three distinct ironsulfur centres. A search for cysteine clusters potentially involved in FeS centre formation revealed neither an exact CX3CPX4CX3C nor CX2DX2CX3C motif; however, one cysteine cluster was found in the
and two in the ß subunit at positions 11201133, 4959 and 97105. When computer-based domain searches were carried out, a GOGAT domain, comprising a putative FMN-binding site and a FeS cluster, was identified at position 8061179, and a glycine-rich region from position 12851422 in the
subunit and two NAD-binding domains at positions 146174 and 279308 in the ß subunit.
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Analysis of gltBD transcription
To study expression of gltB and gltD and its (putative) regulation, total RNA was isolated from C. glutamicum cells before and after nitrogen starvation and hybridized with a digoxigenin-labelled gltB anti-sense RNA probe. While no gltB transcript was detected in cells grown under nitrogen excess, hybridization signals were observed within 15 min after cells were transferred to a nitrogen-free medium (Fig. 2a). These results show that GOGAT is up-regulated on the level of transcription upon nitrogen starvation.
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To determine the transcriptional start site of the gltBD operon, total RNA isolated from nitrogen-starved cells was used as a template in a RACE assay. The transcriptional start site was identified 41 bp upstream of gltB and a -10 and -35 consensus sequence typical for C. glutamicum promoter regions (Patek et al., 1996 ) were identified (Fig. 3
). In addition, an insertion sequence which prefers conserved target sequences located adjacent to genes involved in aspartate and glutamate metabolism, ISCg2, is located 756 bp upstream of gltB (Quast et al., 1999
). The role of this insertion sequence in C. glutamicum is unclear. A putative terminator structure with a free energy of 66·8 kJ mol-1 was identified 16 bp downstream of the gltD stop codon spanning nucleotides 66336663.
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To verify the assumption that AmtR might regulate gltBD expression, Northern hybridization experiments were carried out using RNA isolated from the wild-type and from amtR deletion strain MJ6-18 before and after induction of nitrogen starvation. Deregulated gltB expression was observed in strain MJ6-18, demonstrating the regulatory role of AmtR in the wild-type (Fig. 4a).
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GOGAT activity
GOGAT activities were determined for C. glutamicum wild-type cells grown in different media. No activity was detectable when ammonium was present in high concentrations (100 mM). When cells were incubated for 3 h without nitrogen source, GOGAT activities of approximately 3 mU (mg protein)-1 were determined, which are in accord with data reported previously (Börmann-El Kholy et al., 1993 ).
Analysis of a reporter gene strain
Since the results obtained for GOGAT showed only a low, hardly detectable enzymic activity, we tested, in addition to the GOGAT measurements, the activity of a reporter gene construct to obtain more reliable results. For this purpose, a gltBlacZ translational fusion was constructed and integrated in the C. glutamicum gltB locus. The resulting strain GltBlacZ was tested under different physiological conditions. When this strain was grown under nitrogen surplus (100 mM ) no ß-galactosidase activity was detected in permeabilized cells, while complete lack of nitrogen sources or limiting amounts of ammonium (4 mM), glutamine (4 mM) and urea (2 mM) induced ß-galactosidase. After 3 h incubation, activities of 6·1±0·8 mU (mg dry weight)-1 were determined upon nitrogen starvation, 24·5±1·5 mU (mg dry weight)-1 when cells were grown in the presence of ammonia, 14·8±1·2 mU (mg dry weight)-1 with glutamine as nitrogen source, and 15·0±1·2 mU (mg dry weight)-1 using limiting amounts of urea. These results indicate that synthesis of GOGAT is induced when cells grow in the presence of limiting nitrogen supply.
Analysis of glt mutant strains
To study the physiological role of GOGAT, both a gltB and a gltD insertion mutant were constructed (for overview, see Fig. 1). When transcription of the gltBD operon was tested in these mutants for control, only minor binding of a gltB probe in gltD disruption strain Glt2 was observed, indicating an enhanced degradation of the truncated messenger RNA, whilst no binding was detected when gltB mutant strain Glt1 was analysed (Fig. 5
). As a consequence, GOGAT activity was not detectable in strains Glt1 and Glt2, irrespective of the nitrogen status of the cells (data not shown).
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In other Gram-positive organisms such as Bacillus subtilis, glt gene transcription is under the control of carbon metabolism regulators as well (Faires et al., 1999 ). Therefore, in addition to growth tests with limiting nitrogen sources, growth tests were also performed with various carbon conditions. No growth defects were observed for glt mutant strains in comparison to the wild-type when cells were grown in the presence of acetate, glucose, glutamate, maltose, or sucrose (2% final concentrations; data not shown).
Methylammonium uptake in strains Glt1 and Glt2
In addition to the growth experiments using urea as nitrogen source, which indicated no connection of GOGAT activity and methylammonium/ammonium transport (see above), [14C]methylammonium transport measurements with wild-type cells and glt mutant strains were carried out. In contrast to E. coli, methylammonium uptake was not negatively affected by a gltB or gltD mutation. Both mutant strains showed no methylammonium uptake when grown with high amounts of nitrogen sources (500 mM ammonium), while transport was induced upon nitrogen limitation. After 3 h nitrogen starvation, the wild-type revealed a methylammonium uptake rate of 14·7 nmol min-1 (mg dry weight)-1, strain Glt1 of 38·8 nmol min-1 (mg dry weight)-1, and strain Glt2 of 25·5 nmol min-1 (mg dry weight)-1. Obviously, methylammonium/ammonium uptake is significantly enhanced in the GOGAT-deficient strains in order to circumvent problems arising from the less effective ammonium assimilation.
Effect of GOGAT deficiency on glutamate production
The C. glutamicum wild-type is able to excrete high amounts of glutamic acid under conditions of biotin limitation (Kinoshita et al., 1957 ; Gutmann et al., 1992
). A prerequisite of this excretion is an efficient glutamate synthesis pathway. To investigate whether there is an influence of GOGAT activity on glutamate production, the wild-type ATCC 13032 and glt mutant strains Glt1 and Glt2 were grown under biotin limitation. When glutamate excreted in the medium was measured, no detrimental influence of the gltB and gltD mutation on glutamate production was observed. In all strains comparable amounts of glutamate were determined, e.g. 0·4 µmol (mg dry weight)-1 produced overnight by the wild-type versus 0·4 µmol (mg dry weight)-1 produced by Glt1 and 0·5 µmol (mg dry weight)-1 excreted by strain Glt2. Obviously, GDH activity alone is sufficient for glutamate production in C. glutamicum.
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DISCUSSION |
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The same enzymic pathways, GDH and GS/GOGAT, are used for ammonium assimilation in the Gram-positive soil bacterium C. glutamicum and Gram-negative enterobacteria such as E. coli. However, transcriptional regulation of the corresponding genes differs significantly. For example, gltBD expression in E. coli is influenced by a number of regulators including LRP and CRP (for summary, see Reitzer, 1996 ) and gdhA transcription is repressed moderately by nitrogen limitation (Riba et al., 1988
), while in this study we showed that gltBD transcription is regulated in C. glutamicum via AmtR and previous studies revealed no down-regulation of GDH activity in response to ammonium limitation (Börmann et al., 1992
; Tesch et al., 1998
). Also in comparison to Bacillus subtilis, the model organism for Gram-positive bacteria with low G+C content, assimilation of ammonium is organized differently in the high-G+C-content C. glutamicum. In B. subtilis, ammonium is assimilated exclusively via the GS/GOGAT pathway since GDH is absent in this organism (for review, see Schreier, 1993
). Transcription of the gltAB operon encoding GOGAT requires a specific positive regulator, GltC, and an intact carbon control protein CcpA (Faires et al., 1999
), and is repressed by TnrA (Belitsky et al., 2000
). In summary, the results obtained for C. glutamicum support the statement of Fisher (1999)
that in addition to the paradigm of nitrogen assimilation and regulation in enterobacteria, a number of different mechanisms might be realized in other organisms.
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ACKNOWLEDGEMENTS |
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Received 10 January 2001;
revised 3 July 2001;
accepted 23 July 2001.