Nitrogen assimilation in Corynebacterium diphtheriae: pathways and regulatory cascades

FEMS Microbiology Letters 208 (2002) 287^293

www.fems-microbiology.org

Nitrogen assimilation in Corynebacterium diphtheriae: pathways and regulatory cascades Lars Nolden, Gabriele Beckers, Andreas Burkovski



Institute of Biochemistry, University of Cologny, Zu«lpicher-Str. 47, D-50674 Cologne, Germany Received 27 November 2001; received in revised form 7 January 2002 ; accepted 17 January 2002 First published online 13 February 2002

Abstract Genes encoding proteins for ammonium uptake, assimilation, and the nitrogen regulatory system in Corynebacterium diphtheriae were studied on basis of homology searches using Corynebacterium glutamicum genes as query sequences. Regulation of transcription of these genes in response to nitrogen starvation was analyzed by RNA hybridization experiments and knock-out mutants were generated to verify the function of distinct genes. In this communication, we were able to identify the key components of ammonium assimilation pathways and nitrogen regulation in C. diphtheriae. Moreover, we show in this study that molecular biology methods and vectors developed for C. glutamicum can be applied in C. diphtheriae. The results obtained strengthens the role of C. glutamicum as a model organism for mycolic acids-containing actinomycetes. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diphtheria; Nitrogen control; Corynebacterium diphtheriae; Corynebacterium glutamicum

1. Introduction Corynebacterium diphtheriae, a Gram-positive bacterium belonging to the group of mycolic acid-containing actinomycetes, is an important pathogen, the causative agent of diphtheria. The bacterium colonizes the upper respiratory tract of humans, where it synthesizes and secretes the very potent diphtheria exotoxin. Diphtheria, one of the major causes of morbidity and mortality in the past, seemed nearly eliminated in industrialized countries, thanks to improved hygenic conditions and vaccination programs, when in 1990 a large scale epidemic started in eastern Europe with over 70 000 cases and 4000 deaths reported within a 5-year period [22]. The risk of a pandemic epidemia of diphtheria and the emergence of a rising number of non-toxigenic C. diphtheriae strains in various countries (for example, see [6,9]) has intensi¢ed interest in C. diphtheriae in general and in its virulence factors in particular. Important factors for pathogenity and virulence in different bacteria are enzymes involved in nitrogen metabo-

* Corresponding author. Tel. : +49 (221) 470 6472 ; Fax : +49 (221) 470 5091. E-mail address : [email protected] (A. Burkovski).

lism. A well-known example is the urease of Helicobacter pylori which is essential for colonization of the stomach by this bacterium [3]. In Mycobacterium tuberculosis, which is closely related to C. diphtheriae, glutamine synthetase (GS) activity is signi¢cant for virulence [7,8] and urease was discussed as a critical determinant of host^pathogen interaction [2]. In the last years, we studied uptake of nitrogen sources, ammonium assimilation, and connected regulatory mechanisms in Corynebacterium glutamicum (e.g. [1,10,11, 14,16,20]). This bacterium is applied in fermentation processes on the industrial scale and by use of di¡erent mutant strains not only large amounts of L-glutamate (1 000 000 t/a) but also L-lysine (450 000 t/a) are produced, in addition to smaller amounts of the industrially less-important amino acids L-alanine, L-isoleucine, and L-proline. In contrast to closely related pathogenic species like C. diphtheriae, Mycobacterium leprae, or M. tuberculosis, C. glutamicum is generally recognized as a non-hazardous organism, which is safe to handle. Furthermore, based on its extremely wellinvestigated central metabolism and well-established molecular biology tools, C. glutamicum seems to be suitable as a model organism for high G+C Gram-positive bacteria in general and mycolic acid-containing actinomycetes in particular. In this study, we pro¢ted by the current knowledge

0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 7 4 - 3

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of C. glutamicum nitrogen-assimilating enzymes and control proteins in an attempt to investigate the corresponding genes and proteins of C. diphtheriae. Using this approach we were able to identify key components of nitrogen assimilation and regulation in this important pathogen.

2. Materials and methods 2.1. Bacterial cells and growth Strains and plasmids used in this study are listed in Table 1. Escherichia coli strain JM109, used for cloning steps, was routinely grown at 37‡C in Luria^Bertani medium [17]. C. diphtheriae strains were grown at 37‡C in brain heart infusion (BHI; Difco, Detroit, MI, USA). If appropriate, 25 Wg ml31 (¢nal concentration) kanamycin was added to the di¡erent media. To induce nitrogen starvation cells of the mid-exponential phase were transferred to nitrogen-free minimal medium [20]. 2.2. General molecular biology methods For plasmid isolation, transformation of E. coli cells, and cloning, standard techniques were used [17]. 2.3. Preparation of chromosomal C. diphtheriae DNA Chromosomal DNA was isolated from C. diphtheriae cells according to the protocol developed to isolate C. glutamicum chromosomal DNA [4]. 2.4. Transformation of C. diphtheriae cells Two protocols were applied to prepare electro-competent C. diphtheriae cells, both modi¢cations of the method described for the generation of competent C. glutamicum cells [12]. Either strain DSM 43988 was grown in BHI medium supplemented with 25 g l31 glycine, 4 g l31 isonicotinic acid hydrazide, and 1 ml l31 Tween 80 overnight at a slightly lowered temperature of 30‡C, or only 1 ml l31 Tween 80 was added to the BHI medium and the incubation temperature was lowered to 22‡C. Subsequently, the cells were harvested, washed ¢ve times in ice-cold 10% glycerol, and ¢nally suspended in this solution. After freezing in liquid nitrogen, aliquots were stored at 380‡C until use. For electroporation, competent cells were thawed on ice, 50-Wl aliquots were transferred to 2mm electroporation cuvettes and mixed with plasmid DNA. Electroporation was carried out using a Bio-Rad (Munich, Germany) Gene Pulser apparatus (600 6, 25 WF, 2.5 kV). After electroporation, cells were transferred to 1 ml BHIS medium (BHI plus 9% sorbitol as osmotic protectant), incubated for regeneration at 37‡C for 2 h, and plated out on BHI-medium agar plates containing

10 Wg ml31 kanamycin. For subsequent streak-outs or liquid cultures 25 Wg ml31 kanamycin was used. 2.5. Preparation of C. diphtheriae RNA, construction of RNA probes, and RNA hybridization analyses Total RNA was prepared after disruption of the C. diphtheriae cells by glass beads using the RNeasy Mini kit as recommended by the supplier (Qiagen, Hilden, Germany). The RNA was spotted directly onto nylon membranes using a Schleicher and Schuell (Dassel, Germany) Minifold I Dot Blotter. Hybridization of digoxigenin-labeled RNA probes was detected with a Fuji luminescent image analyzer LAS1000 or Kodak X-OMAT X-ray ¢lms using alkaline phosphatase-conjugated anti-digoxigenin Fab fragments and CSPD as light-emitting substrate as recommended by the supplier (Roche Diagnostics, Mannheim, Germany). For RT-PCR the One-Step RT-PCR kit was used as described (Qiagen, Hilden, Germany). For the synthesis of RNA probes, corresponding DNA fragments were ampli¢ed by PCR using chromosomal DNA as template. The PCR products were ligated to vectors pGEM3z or pGEM4z (Promega, Heidelberg, Germany) and the resulting plasmids (see Table 1) were applied in in vitrotranscription experiments after linearization. 2.6. Directed gene disruptions of C. diphtheriae amtR and glnD For the chromosomal disruption of the C. diphtheriae amtR gene, a 0.5-kb internal DNA fragment was ampli¢ed via PCR using chromosomal DNA of strain DSM 43988 as template and the following primers: 5P-GCGCGCGGATCCCGTGAGGAAATCCTTGATGCCTCAG-3P; 5P-GCGCGCTCTAGAGCTAGAACGGGAAGTTCATCG-3P. Using the BamHI and XbaI sites introduced in via the PCR primers (shown in bold) the DNA fragment was ligated to BamHI/XbaI-restricted and -dephosphorylated pK18mob DNA. The resulting plasmid pK18amtRCD was ampli¢ed in E. coli JM109 and used subsequently for electroporation of C. diphtheriae cells. Kanamycin-resistant C. diphtheriae cells carried the vector integrated via recombination in the chromosomal amtR gene and were designated LNCD -AmtR. PCR for the ampli¢cation of an internal 0.6-kb glnD fragment was carried out using the primers 5P-GCGCGCGAATTCAGCAGATCAAGAGTCAACAGGTC-3P and 5P-GCGCGCTCTAGAGCTACGAGATATTGAGCTGATCAG-3P. After restriction of the PCR product with EcoRI and XbaI (restriction sites introduced via PCR primers, shown in bold) and ligation to correspondingly digested pK18mob DNA, the resulting vector pK18glnDCD was used to generate C. diphtheriae disruption mutant LNCD -GlnD as described above. Gene disruptions were veri¢ed by PCR (data not shown).

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Table 1 Strains and plasmids used in this study Strains/plasmids C. diphtheriae strains DSM 43988 LNCD -AmtR LNCD -GlnD E. coli strains JM109 Plasmids pGEM3z pGEM4z pGEM16S pGEMamtR pGEMgdh pGEMglnA pGEMglnK pK18mob pK18amtRCD pK18glnDCD pZ8-1

Relevant genotype/description

Reference/source

avirulent isolate from throat amtR gene disruption, Kmr glnD gene disruption, Kmr

DSMZ, Braunschweig, Germany this study this study

FP traD36 proAþ Bþ lacIq v(lacZ)M15/v(lac-proAB glnV44 e143 gyrA96 recA1 relA1 endA1 thi hsdR17

[24]

E. coli plasmid for in vitro transcription, Apr E. coli plasmid for in vitro transcription, Apr 0.5-kb internal 16S rRNA gene fragment from C. glutamicum in pGEM3z 0.5-kb internal amtR gene fragment of C. diphtheriae in pGEM3z 0.5-kb internal gdh gene fragment of C. diphtheriae in pGEM3z 0.5-kb internal glnA gene fragment of C. diphtheriae in pGEM3z 0.3-kb internal glnK gene fragment of C. diphtheriae in pGEM4z C. glutamicum gene disruption vector, oriE: coli , Kmr internal 0.5-kb amtR fragment in pK18mob internal 0.9-kb glnD fragment in pK18mob ptac Kmr

Promega, Heidelberg, Germany Promega, Heidelberg, Germany [1] this study this study this study this study [18] this study this study Degussa AG, Halle-Ku«nsebeck, Germany

Apr , resistance to ampicillin ; Kmr , resistance to kanamycin.

2.7. Preparation of cell extracts and determination of GS activity GS activity was determined using the ‘in vivo-like’ method [23]. For this purpose, cell extracts were prepared by ultrasonic cell disruption followed by low-speed centrifugation to remove cell debris. GS activity was determined in the presence of 1 mM MnCl2 by a coupled photometrical test. In the ¢rst reaction step, GS metabolizes glutamate, ammonium, and ATP. ADP generated in this reaction is phosphorylated by pyruvate kinase. In this reaction the donor of the phosphoryl group, phosphoenol pyruvate, is converted to pyruvate which is metabolized by lactate dehydrogenase to lactate. In this reaction, NADH is consumed and the decrease of this compound is measured photometrically. The protein content of samples was determined using a modi¢ed Bradford assay (Roti Nanoquant, Roth, Karlsruhe, Germany).

2.8. Computer-assisted analyses C. diphtheriae sequence raw data generated and made public by the Sanger Centre were screened by the BLASTn (nucleotide vs. nucleotide sequence) and BLASTx (protein vs. translated DNA) program at the Sanger Centre (http:// www.sanger.ac.uk). Sequences extracted from there were further analyzed, e.g. for transmembrane helices, using the TMHMM program (http://www.ebi.ac.uk).

3. Results 3.1. Identi¢cation and expression analyses of C. diphtheriae genes In order to identify genes encoding proteins of C. diphtheriae involved in nitrogen assimilation and nitrogen control the amino acid sequences of C. glutamicum proteins

Table 2 Comparison of C. glutamicum and C. diphtheriae genes and proteins C. glutamicum genes (proteins)

Identity in C. diphtheriae (nucleotide sequence, %)

Identity in C. diphtheriae (amino acid sequence, %)

amt (Amt) amtB (AmtB) amtR (AmtR) gdh (GDH) glnA (GSI) glnA2 (GSIK) glnE (ATase) glnD (UTase) glnK (GlnK) gltBD (GOGAT)

not identi¢ed 62a 69 76 79 70 67 62 74 not identi¢ed

not identi¢ed 50a 70 73 79 78 63 47 73 not identi¢ed

a

Data for amtB/AmtB rely on a truncated open reading frame in the C. diphtheriae database.

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Fig. 2. Transcriptional coupling and expression regulation of C. diphtheriae genes. RT-PCRs were carried out using primers annealing in the putative ¢rst and last gene of the operon using total RNA isolated from cells grown in rich medium (lane 1) or from nitrogen-starved cells (lane 2). DNA marker (M), V-BstEII-digest (from top to bottom 8.4, 7.2, 6.4, 5.7, 4.8, 4.3, 3.7, 2.3, 1.9, 1.4, 1.3, and 0.7 kb). A: glnA2^glnE operon with an expected size of the RT-PCR product of 0.9 kb; B: amtB2^ glnK^glnD operon with an expected size of the RT-PCR product of 1.2 kb. Fig. 1. RNA hybridization experiments. Total RNA isolated from cells grown in rich medium (lane 1) and after 15 (lane 2) and 30 min (lane 3) of nitrogen starvation was hybridized with an amtR, a gdh, glnA, and glnK probe. The 16S rRNA gene was monitored as a control using a corresponding probe designed for the C. glutamicum gene, which exhibited 95% identity.

were used as query sequence to screen the available C. diphtheriae data. These were generated by the C. diphtheriae Sequencing Group at the Sanger Centre and can be obtained from ftp ://ftp.sanger.ac.uk/pub/pathogens/cdip/ CDIP.dbs. The C. diphtheriae NCTC 13129 strain sequenced is a clinical isolate from the United Kingdom representative of an epidemic clone now circulating within eastern Europe. Although the full genome sequence is still being assembled and has not yet been annotated, a signi¢cant number of deduced proteins potentially relevant for nitrogen metabolism were identi¢ed by this approach. In general, a high percentage of identical amino acids was observed, varying between 47 and 79% depending on the di¡erent proteins of C. glutamicum and C. diphtheriae (Ta-

ble 2). The DNA sequences resulting from this data-mining approach were directly used for the analysis of corresponding genes by expression pro¢ling in respect to nitrogen control. In order to investigate transcription of the identi¢ed genes in dependence of the cellular nitrogen status, total RNA was prepared from C. diphtheriae strain DSM 43988 grown in nitrogen-rich medium and from cells after 15 and 30 min of incubation in nitrogen-free medium. In order to generate RNA probes for the di¡erent transcripts, internal DNA fragments of the genes of interest were ampli¢ed via PCR. All primers designed from strain C. diphtheriae NCTC 13129 nucleotide sequences were annealing in these PCR approaches using chromosomal DNA from C. diphtheriae strain DSM 43988 as template. Furthermore, hybridization of RNA probes could be carried out under stringent conditions. These observations demonstrate that genes encoding proteins important for nitrogen metabolism are highly conserved between di¡erent strains of C. diphtheriae and results ob-

Fig. 3. In£uence of site-speci¢c gene disruptions on glnA transcription in C. diphtheriae. Total RNA isolated from cells grown in rich medium (lane 1) and after 15 (lane 2) and 30 min (lane 3) of nitrogen starvation was hybridized with a glnA probe and a 16S rRNA gene probe for control.

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tained on the basis of non-virulent strains which can be handled without unacceptable risk can be transferred to toxigenic strains. 3.2. Ammonium-uptake systems In C. glutamicum, two genes encoding ammonium carriers with di¡erent substrate speci¢city were identi¢ed, designated AmtB and Amt [10,14,20]. When BLAST searches were carried using the C. glutamicum AmtB amino acid sequence, two open reading frames were identi¢ed in the C. diphtheriae genome data bank. Subsequent analyses showed that both sequences encode a small membrane protein (with 2 or 3 transmembrane helices), each revealing high identity to the N-terminal region of C. glutamicum AmtB. Since all AmtB proteins known so far exhibit 10^ 12 predicted transmembrane helices, obviously only a truncated amtB gene is present in the C. diphtheriae data base. An amt gene, encoding a methylammonium/ammonium transporter could not be identi¢ed. The latter result is in accord with the lack of methylammonium uptake observed by transport measurements (data not shown). 3.3. Assimilation of ammonium and regulation of GS activity Many bacteria have two primary pathways to facilitate the incorporation of ammonium into the key nitrogen donors for biosynthetic reactions, glutamate and glutamine. While glutamate dehydrogenase (GDH) is used under high ammonium supply, the glutamine synthetase/glutamate synthase (GS/GOGAT) system allows the assimilation of ammonium when present in the medium at low concentrations (91 mM). However, as deduced from the results of RNA hybridization experiments, transcription of the C. diphtheriae gdh gene is signi¢cantly increased in response to nitrogen shortage (Fig. 1). This is also the case for gdh transcription in C. glutamicum (L. Nolden, unpublished results). The reason(s) for this unexpected and interesting regulation will be addressed in a future project. The glnA gene, encoding GSI, is expressed at a low level in C. diphtheriae cells grown in nitrogen-rich BHI medium, while transcription of this gene is enhanced in response to nitrogen shortage (Fig. 1). In accordance with the expression data, the determination of GS activity revealed an increase from 0.23 þ 0.01 to 0.54 þ 0.01 U (mg protein)31 in response to 2.5 h of nitrogen deprivation. When we examined the deduced amino acid sequence of the C. diphtheriae GSI, we observed an adenylylation motif at amino acid positions 395^413 with a tyrosyl residue at position 406 as the adenylylation site, strongly indicating that activity regulation of GSI occurs. In fact, a glnE gene, encoding adenylyltransferase, was identi¢ed in the C. diphtheriae data base. The genetic organization of glnE is conserved among C. glutamicum and C. diphtheriae. In both organisms, a paralog of the glnA gene, glnA2, which

291

encodes a enzyme of the GSIK group [15], is located directly upstream of glnE. Using primers annealing to the glnA2 and glnE nucleotide sequences in a RT-PCR approach, we obtained a common 0.9-kb product, showing that transcription of glnA2 and glnE is coupled (Fig. 2A). Control reactions without reverse transcriptase carried out to exclude a contamination of the RNA preparations with chromosomal DNA gave no product (data not shown). As in C. glutamicum [15], expression of this operon is not signi¢cantly regulated under the conditions examined (Fig. 2A). The down-regulation of GS activity observed in response to an ammonium pulse (100 mM) within a time period of 25 min was quite low (from 0.54 þ 0.01 to 0.43 þ 0.01 U (mg protein)31 ), indicating either a very slow regulation of GSI activity or high activity of the unregulated GSIK protein under these conditions. An unequivocal identi¢cation of the gltBD operon was not possible on the basis of the present sequence information. While gltB was totally absent, only a small sequence showing homology to part of gltD was observed. Although this DNA fragment showed an expression pattern identical to that of gltD from C. glutamicum [1] when RT-PCR analyses were carried out using RNA isolated from cells grown in BHI medium and incubated in nitrogen-free salt solution (data not shown), without further experimental evidence the signi¢cance of this ¢nding remains unclear. 3.4. Nitrogen signal-transduction cascade Central components of the signal-transduction cascade for nitrogen control in C. glutamicum are encoded by glnK and glnD [16]. Homologs of both genes are present in C. diphtheriae and the deduced amino acid sequence of GlnK revealed a typical uridylylation site, a tyrosyl residue at amino acid position 51. As found in C. glutamicum, GlnK seems to be the only PII -type signal-transduction protein in C. diphtheriae, also. RNA hybridization experiments indicated a slight up-regulation of glnK expression in response to nitrogen starvation, which would be in accordance with results obtained for C. glutamicum [16]. In C. glutamicum, the glnK gene and the glnD gene are organized in an operon together with amtB [11]. While amtB homologs are frequently clustered with glnK homologs in a wide number of Archaea and bacteria [21] amtB^glnK^ glnD clusters were observed in di¡erent actinomycetes [16], e.g. M. tuberculosis and Streptomyces coelicolor. At least for the amtB^glnK^glnD genes of C. glutamicum transcriptional coupling was shown [11]. In RT-PCR experiments with primers annealing in the 3P part of amtB and in the 5P part of glnD, a 1.2-kb DNA fragment was detected, demonstrating an amtB^glnK^glnD operon in C. diphtheriae, also (Fig. 2B) while control reactions without reverse transcriptase carried out to exclude a contamination of the RNA preparations with chromosomal DNA gave no product (data not shown).

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3.5. Regulation of transcription In C. glutamicum nitrogen starvation-dependent transcription of genes is regulated by a global repressor protein designated AmtR [11]. In the C. diphtheriae DNA sequence only one putative homolog of the corresponding C. glutamicum gene was detected, which was, as C. glutamicum amtR, constitutively expressed (Fig. 1). Furthermore, analyses of a corresponding mutant strain (see Section 3.6.) demonstrated that C. diphtheriae amtR encodes a repressor protein essential for nitrogen control. In C. glutamicum the binding motif of the AmtR protein varies from ATCTAT [15] to ATCTATAGN4 ATAGN20 ATCTATAGN4 ATAG [11] making sequence homology searches di⁄cult. However, at least upstream of the glnA gene, at position 3106 to 3101 relative to the start codon, an AmtR-like consensus motif, ATCTAT, was observed by BLAST searches in C. diphtheriae, also. 3.6. Directed inactivation of genes in C. diphtheriae Genetic analysis of C. diphtheriae has long been frustrated by the lack of appropriate genetic tools for this organism and only very recently a system for creating directed gene disruptions was established in C. diphtheriae [19]. Our observation that methods for the isolation of chromosomal DNA and total RNA or for the preparation of competent cells developed for C. glutamicum can be, with minor modi¢cations, applied in C. diphtheriae, also (see Section 2), tempted us to test also C. glutamicum vectors in this organism. In fact, an integration vector developed for gene disruptions in C. glutamicum was successfully applied to generate site-speci¢c mutations of the amtR and glnD gene, respectively, to verify the function of the corresponding proteins in C. diphtheriae. As an example, the transcription of glnA was monitored. In the wild-type, expression of glnA is increased in response to nitrogen starvation (Figs. 1 and 3). This adaptation to nitrogen limitation should be impaired when the signal-transduction cascade of nitrogen control is disrupted. In fact, amtR insertion strain LNCD AmtR revealed a high level constitutive glnA expression, i.e. repression of transcription in nitrogen-rich medium was lost (Fig. 3). This result is in accordance with a repressor function of the AmtR protein. In contrast, glnD mutant LNCD -GlnD had lost its ability to enhance glnA transcription in response to nitrogen shortage (Fig. 3). This strain is unable to sense nitrogen limitation, a result which was also obtained for a corresponding C. glutamicum glnD mutant strain in respect to glnA transcription [15].

diphtheriae using C. glutamicum as a model organism. Deduced from the genetic information for the assimilation of ammonium both the GDH and GS/GOGAT pathway is present in C. diphtheriae. Besides the identity on protein level, a similar pattern of expression of di¡erent genes in response to nitrogen starvation, also the genetic organization of genes encoding key components of nitrogen regulatory cascade are conserved among C. glutamicum and C. diphtheriae. Moreover, also the nitrogen control mechanism on the level of transcription was elucidated for C. diphtheriae. Transcriptional control of genes expressed in response to nitrogen starvation is controlled by a global repressor protein, a mechanism until now solely found in C. glutamicum. The experiments carried out in this study showed that molecular biology methods and vectors developed for C. glutamicum can be applied in C. diphtheriae with only minor modi¢cations. Besides the data presented for the isolation of total RNA or chromosomal DNA, for the preparation of competent cells, and for the use of a gene disruption vector, we were also able to introduce C. glutamicum plasmid pZ8-1 in C. diphtheriae carrying a powerful tac promoter for expression experiments (data not shown). The approach presented here might be also advantageous to investigate nitrogen assimilation and corresponding regulatory mechanisms in other coryneform bacteria. In recent years numerous members of the genus Corynebacterium have been recognized with increasing frequency as important opportunistic human pathogens, especially in immunocompromised patients and patients under intensive care [5]. Some of these pathogenic species, including Corynebacterium jeikeium and Corynebacterium urealyticum, are highly resistant to the majority of clinically relevant antibiotics [5]. Similarly, clinical isolates of Corynebacterium striatum have been characterized by their multiresistance pro¢les upon antimicrobial susceptibility [13]. With a rising number of bacterial genome sequencing projects in progress (for examples see http://www.integratedgenomics.com; http://www. sanger.ac.uk; http://www.tigr.com) it can be expected that also the genomic sequence of these pathogens will be available in the near future. Based on this information, a better understanding of di¡erent coryneform bacteria in respect to physiology and global regulatory networks like nitrogen control might help to reinforce the antibiotic armamentarium against pathogenic strains.

Acknowledgements The authors wish to thank R. Kra«mer for his continuous support and interest.

4. Discussion In this study, we identi¢ed the genes encoding central enzymes for ammonium uptake and assimilation as well as the components of the nitrogen regulatory cascade in C.

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