Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins

FEMS Microbiology Letters 173 (1999) 303^310

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins Marc Jakoby, Reinhard Kraëmer, Andreas Burkovski * Institut fuër Biochemie, Universitaët zu Koëln, Zuëlpicher-Str. 47, D-50674 Cologne, Germany Received 23 November 1998; received in revised form 9 February 1999; accepted 10 February 1999

Abstract The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PII (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Nitrogen control; Gram-positive bacterium; Actinomycete ; Mycobacterium

1. Introduction Corynebacterium glutamicum is used in fermentation processes on an industrial scale: large amounts of glutamate (800 000 t/a) and lysine (250 000 t/a) are produced with di¡erent mutant strains besides smaller amounts of alanine, isoleucine and proline [14]. A crucial step of amino acid production, and of the cellular metabolism in general, is the assimilation of nitrogen and its regulation. These processes have

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

been most extensively studied in Escherichia coli (for reviews, see [16,18]). In this Gram-negative enterobacterium adenylylation regulates the catalytic activity of glutamine synthetase I (GSI), which is encoded by the glnA gene. Covalent binding of AMP to a speci¢c tyrosyl residue of GSI occurs in response to an increase in the intracellular nitrogen availability. This modi¢cation is catalyzed by an adenylyltransferase, encoded by glnE, which is controlled by the PII protein. PII , encoded by glnB, functions as a trimer. Depending on its modi¢cation status, it binds to di¡erent enzymes thereby altering their catalytic activity. When PII is covalently modi¢ed with UMP at a speci¢c tyrosyl residue in each subunit, it

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 0 8 5 - 3

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promotes the deadenylylation of GS by the adenylyltransferase. Vice versa, unuridylylated PII promotes adenylylation of GS, which is inactive in this form. The uridylylation status of PII depends on the activity of UTase, encoded by glnD, the primary nitrogen sensor of the cell. Beside its function in regulating GS enzyme activity, PII also controls transcription of di¡erent genes via the NtrB/NtrC two-component system. Thus, PII is a central signal transduction protein in nitrogen assimilation. Less information is available for the regulation in Gram-positive bacteria. In Bacillus subtilis (for review, see [22]) the glutamine synthetase was studied and found not to be regulated via adenylylation. Although the whole genome of this bacterium was sequenced [13] the exact mechanism of the regulation of nitrogen assimilation has not yet been unraveled. Also in other Gram-positive bacteria such as clostridia and streptomycetes, the components of the nitrogen assimilation regulatory pathway are only partially known [18,22]. Since we are interested in global regulatory mechanisms in C. glutamicum, we started to characterize the signal transduction pathway of nitrogen regulation in this organism.

2. Materials and methods

concentrations. To induce nitrogen starvation, C. glutamicum cells were grown overnight in LB medium and subsequently transferred to minimal medium without nitrogen source [24]. 2.2. Molecular biology techniques For plasmid isolation, transformation and cloning standard techniques were used [20]. Chromosomal DNA from C. glutamicum was isolated according to Eikmanns et al. [6]. Southern blotting was performed using the DIG DNA labeling and detection kit from Boehringer (Mannheim, Germany). Site-directed mutagenesis was carried out using the QuikChange site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). DNA sequence determination was carried out using the BigDye Terminator kit and an ABI prism 310 sequencer (Applied Biosystems, Weiterstadt, Germany). 2.3. Determination of glutamine synthetase activity Cell extracts were prepared by ultrasonic cell disruption followed by low speed centrifugation to remove cell debris. GS activity was determined using the `in vivo-like' test with 1 mM MnCl2 [26].

2.1. Bacterial strains and growth

2.4. Non-denaturing polyacrylamide gel electrophoresis and Western blotting

Strains and plasmids used in this study are listed in Tables 1 and 2. Bacteria were grown in LB medium [20] at 30³C (C. glutamicum) or 37³C (E. coli). If appropriate, antibiotics were added in standard

Modi¢cation of PII was shown by non-denaturing gel electrophoresis [8]. The protein was detected after Western blotting using an antiserum against PII from Synechococcus sp. strain PCC 7942 [7], anti-rabbit

Table 1 Strains used in this study Strain C. glutamicum Amt3 ATCC 13032 MB2897 MJ4-26 MJ5 R127 E. coli DH5Kmcr S17-1

Relevant genotype/description

Reference

ATCC 13032 amt: :pEM1 wild-type R127 glnB: :pK18mob ATCC 13032 vglnA ATCC 13032 glnB: :pK18mob restriction-de¢cient mutant strain

[24] [1] This study [12] This study [15]

endA1 supE44 thi-1 V3 recA1 gyrA96 relA1 deoR v(lacZYA-argF) U169 x80vlacZ vM15mcrA v(mmr hsdRMS mcrBC) thi-1 F3 endA1 hsdR17 supE44 V3 pro

[9]

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[25]

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Table 2 Plasmids used in this study Plasmid

Description

Reference

pAmt-H3 pEM1 pJC1 pJCglnA pJCY405F pINS-PII pK18mob pPII-333 pPII-H3 pPII-Sfu pUC18 pUCamtP

pEM1 carrying a HindIII fragment downstream of amt KmR , oriT E. coli/C. glutamicum shuttle vector, KmR , oriVE:c: , oriC:g: pJC1 carrying the glnA wild-type allele pJC1 carrying the glnA (Tyr405Phe) allele pK18mob carrying a 165-bp glnB fragment KmR , oriV oriT mob pK18mob carrying the complete glnB gene pK18mob carrying a 3.3-kb HindIII fragment (glnB glnD srpP) pK18mob carrying a 20-kb SfuI fragment with glnB upstream region plac, ApR pUC18 carrying a 2.4-kb BamHI insert (PftsY amtP glnB)

This [23] [3] This This This [21] This This This [27] This

R

study

study study study study study study study

R

Ap , resistance to ampicillin ; Km , resistance to kanamycin.

alkaline phosphatase conjugate and BCIP/NBT tablets as substrate (Sigma, Deisenhofen, Germany). 2.5. Computer-assisted sequence analysis and nucleotide sequence accession numbers For nucleotide sequence analysis the EMBL (Heidelberg, Germany) data bank program BLASTX was used. Protein sequence alignments and protein secondary structure analyses were carried out using the PDHhtm program (EMBL, Heidelberg, Germany). The DNA sequences generated in this study were submitted to GenBank (EMBL, Heidelberg, Germany, accession numbers AJ007732 (amt cluster) and AJ010319 (glnB cluster)).

not phosphorylation is sensitive to the addition of this enzyme. Additionally, a glnA mutant was constructed via site-directed mutagenesis in which the putative adenylylation site Tyr405 was replaced by a phenylalanyl residue (Y405F). When GSI activity was tested in vitro, the wild-type enzyme showed high activity after N starvation, which decreased

3. Results and discussion 3.1. Regulation of GSI activity In bacteria, the central enzyme for the assimilation of ammonium under limiting conditions is glutamine synthetase. Recently, we isolated the corynebacterial glnA gene, encoding glutamine synthetase I (GSI) [12]. When the DNA sequence was analyzed, a typical adenylylation site, Tyr405, was identi¢ed. Since we were interested to know whether the regulation of this central enzyme of nitrogen assimilation occurs via adenylylation, we studied the e¡ect of snake venom phosphodiesterase on cell extracts of C. glutamicum (Fig. 1). Adenylylation (and uridylylation) but

Fig. 1. Glutamine synthetase activity depending on snake venom phosphodiesterase treatment. Cells of glnA deletion strain MJ426 carrying plasmid pJCglnA (A) and pJCY405F (B) were starved of nitrogen for 3 h to induce GSI synthesis. 30 min prior to cell disruption no NH4 Cl (1) or 20 mM NH4 Cl was added (2^4) to the culture. GS activity of the cell extracts was determined directly (1, 2) and after 15 min of incubation at 37³C with the addition of 100 Wg ml31 (¢nal concentration) snake venom phosphodiesterase (3) or without as a control (4). The experiments were carried out in triplicate and standard errors are indicated.

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upon NH4 Cl addition and was restored by treatment with snake venom phosphodiesterase. In contrast, the activity of the Y405F mutant was not downregulated upon the addition of NH4 Cl and, as a consequence, treatment with phosphodiesterase had no signi¢cant in£uence (Fig. 1). From these results it was concluded that GSI is regulated depending on the nitrogen status of the cell and that Tyr405 is the crucial amino acid residue for this regulation, which most likely occurs via adenylylation. Interestingly, upon NH‡ 4 pulse, GS activity is downregulated in 30 min only to 40%. If this observation is the result of a low adenylyltransferase activity or the existence of an additional GS enzyme as found in Streptomyces viridochromogenes [11] remains unclear. The corynebacterial mechanism of GSI regulation, which is in accord with previously published studies on GS gene evolution [19], is similar to that of the E. coli enzyme, a result which prompted us to investigate the existence of further genes and proteins homologous to components of the E. coli regulatory cascade in C. glutamicum.

Data base searches revealed two open reading frames downstream of amt (Fig. 2A): an ocd-like gene, encoding a putative ornithine cyclodeamidase (32% identity with Sinorhizobium meliloti Ocd; SwissProt P33728) and the 5P part of a soxA gene (33% identity with Streptomyces sp. SoxA; SwissProt S39464), encoding a putative sarcosine oxidase. Since the glnB gene was found not to be clustered with the amt gene in C. glutamicum, we carried out a PCR screening with degenerated primers deduced from the Mycobacterium tuberculosis glnB sequence. Using Taq polymerase as recommended (Boehringer Mannheim, Germany), chromosomal DNA isolated from strain ATCC 13032 as template, 10 WM of primer A (sense, 5P-CA(AG) AA(AG) GG(ACGT) CA(CT) AC(ACGT) G-3P), 5 WM of primer B (antisense, 5P-AAC CA(ACGT) AC(CT) TT(ACGT) CC(AG) TC-3P) and 35 cycles of 0.5 min 94³C, 1.0 min 42³C, 1.0 min 72³C, a 165-bp DNA fragment was isolated. Sequence analysis of this PCR product revealed high similarity to the M. tuberculosis glnB gene (data not shown).

3.2. Strategies for identi¢cation of the glnB gene

3.3. Isolation and sequence of the glnB gene

In E. coli the signal transduction protein PII , encoded by glnB, plays a pivotal role in the regulation of nitrogen assimilation (see Section 1). We therefore focused our e¡orts on the isolation and sequencing of glnB. We tried to take advantage of the fact that in several organisms (e.g. Azospirillum brasiliense, B. subtilis, E. coli) glnB, or its corresponding paralog glnK or glnZ, is located adjacent to the amt gene, which encodes an ammonium uptake system. Since in C. glutamicum the ppc gene [5] and a secG homolog (Jakoby, unpublished) are located upstream of amt, we sequenced its downstream DNA region. For this purpose, a plasmid rescue with the amt disruption mutant Amt3 [24] was carried out. Chromosomal DNA of this strain was isolated and digested with HindIII, which cuts only once in the construct used for gene disruption. After religation and rescue of insertion vector pEM1 with part of the chromosomal DNA, a SacI fragment carrying the amt gene and its adjacent downstream region was identi¢ed by Southern blotting (data not shown) with an 0.5-kb AvaII/PvuII amt probe (Fig. 2A). This fragment was ligated to SacI-cleaved pUC18 DNA and sequenced.

As a ¢rst step to isolate the complete glnB gene an insertion mutant was constructed using the 165-bp PCR product ligated to insertion vector pK18mob. Unfortunately, a small fragment size dramatically decreases the e¤ciency of homologous recombination in C. glutamicum and as a consequence we were not able to achieve a glnB disruption in wildtype strain ATCC 13032. To improve the e¤ciency of recombination, we used the restriction-de¢cient strain R127 as a host, which is not able to degrade the DNA of the insertion vector. As a consequence the likelihood of recombination is improved and we were successful in constructing a glnB insertion strain, designated MB2897. In a second step, chromosomal DNA of the insertion mutant was isolated and treated with either KpnI or HindIII. Both enzymes cut only once in the insertion construct and as a consequence successful rescue of the plasmid is only achieved if adjacent chromosomal DNA is coisolated. After ligation of the DNA cut with HindIII or KpnI, transformation of E. coli and screening for kanamycin-resistant clones the adjacent chromosomal DNA was analyzed and a complete glnB se-

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Fig. 2. Isolated C. glutamicum gene clusters. A : Organization of the amt region. B: Organization of the glnB gene cluster. The amt probe for Southern blotting (gray bar) and the site of vector insertion in glnB are shown. T indicates a rho-independent terminator structure. Restriction enzyme recognition sequences are indicated as follows : B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NruI; P, PstI ; Sf, SfuI. Dotted line : not drawn to scale.

quence was obtained. In order to verify this nucleotide sequence in the wild-type strain, the complete glnB gene was cloned in pK18mob (pPII-333). This larger insert was now used for integration in ATCC 13032, leading to strain MJ5. Chromosomal DNA of MJ5 was restricted with HindIII and the resulting religation products were analyzed. A recombinant plasmid with a 3.3-kb insert of chromosomal DNA (pPII-H3) carried the complete glnB gene, which en-

codes a 112-amino acid protein with high sequence similarity to other known PII proteins. With the M. tuberculosis PII (glnB gene product) 68% identical amino acids were found, with PII from E. coli (glnK) and A. brasiliense (glnB) 58%. As deduced from the nucleotide sequence, which was identical for R127 and ATCC 13032, PII has a calculated molecular mass of 12.2 kDa and a pI of 5.0. A typical uridylylation site, a tyrosyl residue at position 51,

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was identi¢ed. With a G+C content of 53% glnB exhibits a DNA composition typical for C. glutamicum [17]. A Southern blotting approach using the glnB PCR fragment as a probe revealed no paralog of glnB in the C. glutamicum genome (data not shown). This situation di¡ers from that in E. coli [10] or A. brasiliense [28] where two PII -encoding genes were found and is similar to that in B. subtilis [13] and M. tuberculosis [2] with a single PII gene. 3.4. Isolation and sequence of the glnB gene cluster In order to analyze also the glnB upstream sequence, another plasmid rescue experiment was carried out: chromosomal DNA of strain MJ5 was digested with SfuI and after religation a 20-kb pK18mob clone, pPII-Sfu, was isolated. Sequence analyses of a 2.4-kb BamHI subclone in pUC18 revealed that glnB is £anked in the 5P direction by an amt paralog (amtP). Additionally, we were able to identify an ftsY gene, putatively involved in protein secretion, upstream of amtP (Fig. 2B). When the nucleotide sequence downstream of the glnB gene on plasmid pPII-H3 was determined, two open reading frames were identi¢ed, namely the complete glnD sequence and a truncated srp gene, encoding a putative signal recognition particle involved in protein secretion (Fig. 2B). The glnD gene, encoding the uridylylating/deuridylylating enzyme (UTase), is characterized by a G+C content of 57%. The deduced protein comprises only 692 amino acids and is signi¢cantly smaller than other known UTases, e.g. the M. tuberculosis glnD gene product with 808 amino acids. It exhibits 35% identical amino acids with the M. tuberculosis UTase.

protein reacted with C. glutamicum cell extracts. C. glutamicum wild-type cells were grown under di¡erent nitrogen supply conditions and cell extracts were subjected to non-denaturing gel electrophoresis. In this gel system uridylylation of PII results in a higher electrophoretic motility compared to the unuridylylated form. A typical shift in the pattern was observed when ammonium was added to nitrogenstarved cells. In this case the completely uridylylated trimeric PII protein (PII 3 ) is stepwise deuridylylated from the PII 3 form with high electrophoretic motility to the PII 0 protein with lower motility [7]. For the C. glutamicum PII protein a similar pattern was detected: after nitrogen starvation three bands were observed, PII 0 , PII 1 and PII 2 (Fig. 3), sometimes also a fourth one with higher electrophoretic motility (PII 3 ) appeared. Upon addition of NH4 Cl the bands with higher motility disappeared. The observed modi¢cation was also removed by snake venom phosphodiesterase (Fig. 3), further arguing for a uridylylation, while phosphorylation as in Synechococcus [7] could be ruled out. 3.6. Characterization of a PII 3 strain When testing the antisera against PII we found that strain R127 reveals a PII 3 phenotype (data not shown), although it has an intact glnB gene (see above). Obviously, during mutagenesis this strain was altered not only in the restriction system but also in PII synthesis or degradation. As a conse-

3.5. Modi¢cation of PII In enteric bacteria, the UTase is the primary nitrogen sensor [4], which covalently modi¢es PII by uridylylation under conditions of nitrogen limitation. As an approach to test modi¢cation of PII , we applied non-denaturing polyacrylamide gel electrophoresis followed by Western blotting. While we did not ¢nd a cross-reaction of an antiserum against E. coli PII with the C. glutamicum protein, a PII -speci¢c antiserum against the corresponding Synechococcus

Fig. 3. Modi¢cation of PII in C. glutamicum. Cells of C. glutamicum wild-type ATCC 13032 were grown under di¡erent N supply conditions, harvested and disrupted by ultrasonic treatment. After centrifugation to remove cell debris and membranes (30 min 100 000Ug) the gel was loaded (30 Wg per lane) as follows : (1) 3 h of N starvation, (2) N-starved plus 20 mM NH4 Cl 30 min before cell disruption, (3) N-starved plus 20 mM NH4 Cl 60 min before cell disruption, (4) extract of N-starved cells treated with snake venom phosphodiesterase.

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work was supported by the Fonds der Chemischen Industrie.

References

Fig. 4. Glutamine synthetase activity of (1) wild-type and (2) strain R127 (PII 3 ). Cells were starved of nitrogen for 3 h to induce GSI synthesis. 30 min prior to cell disruption no NH4 Cl (A) or 20 mM NH4 Cl was added (B) to the culture. The experiments were carried out in triplicate and standard errors are indicated.

quence, in R127 GSI activity is in contrast to the wild-type not downregulated upon NH‡ 4 pulse (Fig. 4). Various attempts to construct de¢ned glnB and glnD deletions in the wild-type failed and an obtained glnD insertion mutant was unstable indicating an essential role of these genes in C. glutamicum. 3.7. Conclusions Based on the results obtained in this study the mechanism of N regulation in the Gram-positive C. glutamicum resembles that in the Gram-negative E. coli and not that of the Gram-positive model organism B. subtilis. Since similar genes encoding proteins with an amino acid identity of 35^68% compared to those of C. glutamicum are annotated in the M. tuberculosis genomic sequence this type of regulation most likely also holds true for this important pathogen.

Acknowledgments Antisera against PII were kindly provided by H.V. Westerho¡ (Amsterdam, The Netherlands) and K. Forchhammer (Munich, Germany). The help of K. Forchhammer to establish the non-denaturing gel electrophoresis is gratefully acknowledged. This

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