Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1
Author for correspondence: A. Engels. Tel: +49 7071 29 76157. Fax: +49 7071 29 5979. e-mail: ae{at}biotech.uni-tuebingen.de
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ABSTRACT |
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Keywords: Streptomyces coelicolor, nitrogen metabolism, adenylyltransferase, glutamine synthetase
Abbreviations: GS, glutamine synthetase; SVPDE, snake venom phosphodiesterase
The EMBL accession number of the internal Streptomyces coelicolor glnE fragment is Y17736.
a Present address: Genetics Department, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
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INTRODUCTION |
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Like enteric bacteria, Gram-positive bacilli contain one GS (for review see Schreier, 1993 ). However, Bacillus subtilis possesses no homologues of the ntr genes from enteric bacteria (Kunst et al., 1997
), and the GS enzyme is not regulated post-translationally by adenylylation (Schreier et al., 1985
). Transcription of glnA is negatively controlled in response to the nitrogen status of the cell by two transcription factors, GlnR (Schreier et al., 1989
) and TnrA (Wray et al., 1996
).
In contrast to other Gram-positive prokaryotes, streptomycetes contain at least two distinct GS enzymes (Behrmann et al., 1990 ; Kumada et al., 1990
): GSI (encoded by glnA; Wray & Fisher, 1988
) is composed of 12 identical subunits and is similar to other bacterial GSs; the heat-sensitive GSII (encoded by glnII; Hillemann et al., 1993
) is an octamer and resembles eukaryotic GSs. Little is known about the function and regulation of the two GS enzymes in streptomycetes. Wray & Fisher (1993
) isolated a regulatory gene (glnR) by complementation of a glutamine auxotrophic Streptomyces coelicolor mutant. GlnR might represent a positive transcription factor for both glnA and glnII (Merrick & Edwards, 1995
). A residue corresponding to Tyr-398, the site of reversible covalent GSI modification in E. coli, is not present in the GSII sequence. Therefore, it was assumed that, as expected for the eukaryote GS type, GSII from streptomycetes cannot be modified post-translationally (Behrmann et al., 1990
). In contrast to glnII, the deduced amino acid sequence of the S. coelicolor glnA gene (Wray & Fisher, 1988
) contained the conserved tyrosyl residue at position 397. Physiological evidence for post-translational GSI modification was obtained for Streptomyces cattleya (Streicher & Tyler, 1981
), S. coelicolor (Fisher & Wray, 1989
) and Streptomyces viridochromogenes (Hillemann et al., 1993
). Grown on poor nitrogen sources, the GSI activity of these strains decreased after addition of ammonium. In each case, the GSI activity was reactivated by snake venom phosphodiesterase (SVPDE) treatment. However, although in vitro ADP-ribosylation was reported for the Streptomyces griseus GSI (Penyige et al., 1994
), the physiological GSI modification pathway in streptomycetes is still unknown. Fisher & Wray (1989
) suggested that, as in enteric bacteria, glutamine (or a metabolite derived from glutamine) is involved in the modification of the S. coelicolor GSI. Interestingly, GSI sequences from streptomycetes show stronger similarity to the GSI proteins from Gram-negative bacteria than to GSI proteins from Gram-positive, sporulating bacteria (Kumada et al., 1993
).
In this paper, we report the identification of glnE from S. coelicolor A3(2), encoding an adenylyltransferase, and we also provide evidence that GlnE functions as a GSI-modifying enzyme.
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METHODS |
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Escherichia coli was cultivated at 37 °C in LB medium or on LB agar (Miller, 1972 ). Ampicillin or kanamycin was added at a concentration of 150 or 50 µg ml-1, respectively.
Molecular cloning.
Plasmid and chromosomal DNA of Streptomyces or E. coli were isolated by established techniques (Hopwood et al., 1985 ; Sambrook et al., 1989
). Procedures for forming and regenerating S. coelicolor protoplasts were carried out as described by Hopwood et al. (1985
). The enzymes used in this study were purchased from Boehringer, New England Biolabs or Pharmacia, and used as recommended by the manufacturer. Hybridization experiments were carried out according to Southern (1975
).
PCR.
The oligonucleotide primers used (Table 1) were synthesized by MWG Biotech. For PCR, the reaction mixture used was: 0·2 µg S. coelicolor chromosomal DNA as template, 1·0 µM of each primer, 10 µl 10x reaction buffer (with 20 mM MgCl2), 5·0% DMSO, 0·2 mM dNTPs, 1·0 µl Taq polymerase (Qiagen). The reaction mixture was overlaid with 60 µl mineral oil. An initial denaturation (2 min, 94 °C) was followed by 35 cycles of amplification (1 min at 92 °C, 2 min at 60 °C, 2·5 min at 72 °C) in a robocycler (Stratagene). PCR products were separated electrophoretically in a 1% agarose gel, isolated by elution (Qiaquick; Qiagen) and cloned directly.
DNA analysis and sequencing.
The PCR-generated glnE fragment cloned in pBluescript II SK(+) (resulting in pDOL15) was reisolated by digestion with EcoRI/BamHI and purified from a preparative gel. By a Klenow fill-in reaction, blunt ends were created for ligation into the HincII site of a pBluescript II SK(+) vector. Two constructs with inserts in opposite orientations were identified by restriction analysis and designated pND4 and pND7 (Table 1). Nested deletions from pND4 and pND7 were generated using the Double-stranded Nested Deletion Kit from Pharmacia, according to the manufacturers recommendations.
The DNA sequence of the cloned 1·5 kb PCR fragment was determined by a standard protocol modified for double-stranded DNA, using the AutoRead Sequencing Kit (Pharmacia) for sequencing on an ALF Sequencer (Pharmacia). Nucleic acid sequences were analysed using the software packages Staden (Staden & McLachlan, 1982 ), blast (Gish & States, 1993
; Altschul et al., 1990
), fasta (Pearson & Lipman, 1988
) and clustal w (Thompson et al., 1994
). ORF analysis was based on the specific codon usage of streptomycetes (Wright & Bibb, 1992
).
Construction of the S. coelicolor mutant E4 and HT107.
For the construction of a glnE replacement plasmid, a kanamycin-resistance cassette (aphII) was ligated as a SmaIHincII fragment (from pUC19aphII) into BstEII-restricted and Klenow filled-in pDOL15. From the resulting plasmid pDOF4, a 3000 bp EcoRISpeI fragment containing the disrupted glnE fragment was ligated into EcoRI/XbaI-restricted pWHM3 (Vara et al., 1989 ), which can be used as a suicide vector in many Streptomyces species (Bruntner & Bormann, 1998
). The resulting replacement plasmid was designated pDOL4.
For the construction of a glnA replacement plasmid, a 1562 bp SmaI fragment from pSF205 carrying the complete glnA gene was ligated into EcoRI-restricted and Klenow filled-in pWHM3 (pDFG1). glnA was inactivated by a hygromycin-resistance cassette (hygB) which was ligated as a 1·7 kb BglII fragment (from pIJ963) into the single BglII site of pDFG1. This plasmid was designated pDFH107.
The S. coelicolor glnE and glnA single knock-out mutants were constructed using the same mutagenesis protocol: the replacement plasmid (pDOL4 or pDFH107) was used to transform E. coli ET 12567 and reisolated. After alkaline denaturation (Oh & Chater, 1997 ), 1 µg plasmid DNA was introduced into S. coelicolor protoplasts via PEG-mediated transformation (Hopwood et al., 1985
). Several thiostrepton-resistant colonies were obtained on R2YE media which had been overlaid with 3 ml NB soft agar (Shirahama et al., 1981
) containing 25 µg thiostrepton ml-1. They were allowed to undergo two rounds of sporulation in the absence of antibiotic and the resulting spores were replica-plated on HA media containing 25 µg kanamycin ml-1 (for selection of glnE mutants) or 25 µg hygromycin ml-1 (for selection of glnA mutants).
Cell harvesting, breakage and crude extract preparation.
In general, S. coelicolor was grown for 2 d. Mycelium was homogenized, harvested by centrifugation, washed twice with disruption buffer (50 mM imidazole, pH 7·0; 150 mM NaCl; 1 mM MnCl2; 0·5 mM DTT) and resuspended in the same buffer. S. coelicolor cells were broken by two consecutive passages of the mycelium through a French press (American Instruments) at 1000 p.s.i. (6900 kPa). Cell debris and membrane fractions were separated from the soluble fraction by centrifugation (20 min, 13000 g) and the supernatant was used as crude extract. Total protein was determined according to Smith et al. (1985 ).
GS assay.
GS was assayed by the -glutamyltransferase method adapted from Shapiro & Stadtman (1970
). The activity of the heat-stable GSI was determined after heat treatment of crude mycelium extracts at 60 °C for 10 min prior to the enzyme assay (Edmands et al., 1987
). Modification of GSI was examined as described by Braña et al. (1986
). However, crude extracts were used instead of whole cells. Reactivation of GS in crude extracts was monitored in 20 mM imidazole/HCl, pH 7·0, containing 2·5 mM MnCl2 at 37 °C. The extracts were incubated at 37 °C for 20 min with SVPDE (Boehringer) at 100 µg ml-1.
Determination of glutamate and glutamine concentrations.
For measuring the glutamate and glutamine pools in S. coelicolor cell extracts, amino acids were derivatized with o-phthaldialdehyde (OPA) (Lindroth & Mopper, 1979 ). Derivatives were loaded on a reverse-phase column (4·6x125 mm; Shandon Hypersil ODS) with 4 µl OPA+2 µl sample+4 µl OPA, and separated by HPLC using a linear gradient of elution buffer A (12·5 mM sodium phosphate buffer, pH 7·3; 0·5% tetrahydrofurane) and elution buffer B (20% elution buffer A, 40% methanol, 40% acetonitrile). Components were detected by their UV absorbance at 340 nm (Liquid chromatograph: HP 1090M, built-in diode array detector, autosampler, thermostability controlled column compartment; detection: Pascal Workstation HP 79994B). Identities of glutamate and glutamine were confirmed by co-migration with standards.
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RESULTS AND DISCUSSION |
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From all primers tested, only the combinations P1/P5 and P1/P3 (see Table 1 and Fig. 3a
) resulted in fragments of the expected size of 1500 bp and 1300 bp, respectively.
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Comparison of the deduced amino acid sequence with protein sequences in the Protein Information Resource database yielded significant similarities to enzymes belonging to the group of nucleotidyltransferases, especially to GlnE proteins (Table 2). A significant similarity (39%) was found to the sequence of the E. coli GlnE, which is the covalent modifier of GSI (van Heeswijk et al., 1993
). Similarities to GlnE proteins are distributed over the whole 1500 bp fragment, which furthermore contained the conserved amino acid sequence (Fig. 1
) assumed to be the functional adenylylation motif in the E. coli enzyme (Holm & Sander, 1995
; Jaggi et al., 1997
). Identification of a glnE gene from S. coelicolor is the first example of an Ntr-like component in a Gram-positive, sporulating bacterium.
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Further hybridization studies determined whether the presence of glnE is a unique feature of S. coelicolor. Using the glnE fragment from S. coelicolor as a probe, the genomes of 18 Streptomyces strains were examined for the presence of hybridizing fragments. The strong signals obtained in all strains (Fig. 2) indicated that glnE is ubiquitous in Streptomyces.
Inactivation of the S. coelicolor glnE gene
To construct a glnE mutant of S. coelicolor the replacement plasmid pDOL4 (Table 1) was introduced into S. coelicolor. Resistance to kanamycin, conferred by the cassettes aphII gene, was used to select transformants in which integration had occurred via a single cross-over.
Putative gene replacement mutants were then screened for by selecting disruption mutants that had lost the ability to grow on thiostrepton-containing medium. Since thiostrepton resistance is mediated by the vector, its loss indicates a second cross-over event. Of 117 kanamycin-resistant colonies obtained by transformation of S. coelicolor with pDOL4 and two subsequent rounds of sporulation, 29 colonies were thiostrepton-sensitive. This implies a frequency of about 25% for the double cross-over event.
To characterize the genotype of the mutants (Fig. 3a), chromosomal DNA from four potential gene replacement mutants was analysed by PCR and Southern hybridization experiments. All mutants possessed the genotype shown in Fig. 3(b, c
) for the S. coelicolor glnE mutant E4 (E4). The fragments amplified in PCR experiments from E4 with primers P1 and P5 were about 2·8 kb in size, whereas a 1·5 kb fragment was obtained with the wild-type (Fig. 3b
). The 1·3 kb size difference corresponds to the size of the inserted kanamycin-resistance cassette. Southern blot analysis with the 1·5 kb glnE fragment (Fig. 3c
) and the 1·3 kb aphII resistance cassette (data not shown) as a probe confirmed this result and indicated that only one copy of the antibiotic-resistance cassette is present in the E4 mutant.
Inactivation of the S. coelicolor glnA gene
From enteric bacteria it is known that GSI (encoded by glnA) is the target of the adenylyltransferase. For the investigation of GlnE function it was, therefore, of interest to have an S. coelicolor glnA mutant. Such a mutant was constructed in the same way as described for the glnE mutant (for details see Methods). From 200 kanamycin-resistant colonies obtained, 15 colonies (7·5%) were thiostrepton-sensitive.
The occurrence of a gene replacement event in the S. coelicolor glnA mutant HT107 was verified by Southern hybridization (data not shown) using a glnA probe (1·8 kb SmaI fragment from pSF205). As indicated in Fig. 4, a single hybridization signal was observed with SmaI-digested wild-type DNA. In contrast, two signals (2·35 and 0·9 kb) were obtained with genomic DNA of HT107. This pattern was expected since the inserted hyg cassette carries additional SmaI sites. This confirmed that the native glnA in the S. coelicolor HT107 mutant was disrupted by insertion of the hygromycin-resistance cassette.
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Salmonella typhimurium glnE mutants have a typical Ntr minus phenotype characterized by reduced growth on poor nitrogen sources (Kustu et al., 1984 ). B. subtilis glnA mutants with low residual GS activity require a high glutamine concentration to sporulate (Fisher & Sonenshein, 1977
). The observation that neither the S. coelicolor E4 nor the HT107 mutant suffered from growth defects may reflect the phenomenon of differently regulated GS enzymes in S. coelicolor, which complement each other under various culture conditions.
The glnE mutant has lost the ability for covalent GSI modification by adenylylation
In enteric bacteria, adenylylation of GSI occurs when the cell senses excess nitrogen, i.e. when is available after growth under nitrogen-limited conditions. Therefore, S. coelicolor wild-type and E4 mutant cells, growing in minimal medium with asparagine as a sole and poor nitrogen source, were subjected to an ammonium shock (see legend of Fig. 5
), and the heat-stable GSI activity in crude mycelium extracts was determined.
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In contrast to the wild-type, no GSI activity was observed in the glnA mutant HT107.
In the E4 mutant, the ammonium upshift resulted in a slight increase in GSI activity and was not affected by SVPDE treatment (Fig. 5). At 2, 5 and 10 min after the shift, GSI activity was about 115% of the pre-shift value. After 20 min a further slight increase was observed.
Adenylylation of GSI after administering an ammonium shock to cells growing in a nitrogen-limited environment has been detected in a number of Gram-negative prokaryotes (Merrick & Edwards, 1995 ). In Gram-positive streptomycetes, GSI modification by adenylylation (Streicher & Tyler, 1981
; Bascarán et al., 1989
; Fisher & Wray, 1989
; Hillemann et al., 1993
) as well as by ribosylylation (Penyige et al., 1994
) had been proposed. The results obtained with the E4 mutant demonstrate for the first time that in S. coelicolor a GlnE-mediated GSI modification takes place. Moreover, the results obtained after SVPDE treatment confirm that GSI is most likely modified by adenylylation in S. coelicolor, since only adenylylation and uridylylation (the latter has never been reported for any GS enzyme), but not ribosylation or phosphorylation, are sensitive to the action of SVPDE.
The results for the glnA mutant HT107 demonstrate that the heat-stable GS activity measured under the conditions used is due to GlnA alone. There is evidence for a putative third GS gene in S. coelicolor (localized on cosmid SCI35; accession no. 3581868) from the S. coelicolor genome project (Sanger Institute, Cambridge). This GS shows more similarity to the type I (36 and 37% similarity to glnA of E. coli and S. coelicolor, respectively) than to the type II GS proteins (20% similarity to glnII of S. viridochromogenes). If this putative additional GS gene is functional in S. coelicolor, the activity is not detectable under our growth and assay conditions.
The decrease in GSI activity (about 40%) in ammonium-shocked S. coelicolor cells is rather low compared to the almost complete inactivation observed for E. coli or cyanobacteria (Stadtman et al., 1980 ; Merida et al., 1991
). This may be due to the tendency of S. coelicolor to grow in mycelial clusters and not as finely dispersed cells. The
shock may be much weaker for Streptomyces cells growing at the centre of a mycelial cluster. As a consequence, the GSI value detected after the shift would be the mean activity of modified GSI from cells growing at the periphery and of unmodified GSI from cells growing at the centre of a cluster. Another reasonable explanation for the incomplete GSI inactivation in S. coelicolor after
shock is that glnE expression or GlnE activity in S. coelicolor is just lower than in enteric bacteria.
Imbalance in the glutamine:glutamate ratio of the E4 mutant
It has been postulated that adenylylation of the GSI enzyme in enteric bacteria is a short-term protection of the intracellular glutamate pool under conditions of sudden ammonium excess (Kustu et al., 1984 ). To test this hypothesis for S. coelicolor, the internal glutamate and glutamine concentrations before and after an ammonium upshift were measured in extracts from both wild-type and glnE mutant cells.
In wild-type cells, the glutamate and glutamine concentrations increased in the first 10 min after the upshift and declined thereafter (Fig. 6). In contrast, the glutamate pool of the E4 mutant decreased in the first 5 min after the ammonium upshift and then increased, reaching approximately 71% of the original value after 20 min. A similar course was observed for the glutamine pool. However, the final glutamine concentration after 20 min was 143% of the original value.
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In similar experiments with glnE mutants from Sal. typhimurium (Kustu et al., 1984 ), the glutamate pool decreased to 10% of the pre-shift value and the glutamine pool increased to 180 times the pre-shift value within 30 min after an ammonium upshift. The much weaker effects in the S. coelicolor glnE mutant may be the result of additional regulatory mechanisms that counterbalance the glutamine:glutamate ratio in S. coelicolor.
Concluding remarks
The glnE mutant described here is the first mutant of an Ntr-like gene in a Gram-positive bacterium. Characterization of the E4 mutant demonstrated that GSI in S. coelicolor is affected by the glnE gene product, possibly by adenylylation (suggested by the effect of SVPDE treatment).
Although a further GSI-like gene was discovered during the S. coelicolor genome sequencing project at the Sanger Institute (Cambridge), the properties of the glnA mutant HT107 show that only GlnA represents the heat-stable GS activity. It cannot be excluded, however, that under certain conditions further GS enzymes are active in streptomycetes. It will, therefore, be interesting to isolate and investigate additional compounds of nitrogen metabolism and its regulatory cascade. Elucidation of this network may provide a basic understanding of the complex mechanisms controlling nitrogen metabolism and, in particular, of its involvement in secondary metabolism.
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ACKNOWLEDGEMENTS |
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This work was supported by the European Union (BIO4-CT95-0198) and by the Bundesministerium für Bildung und Wissenschaft, Forschung und Technologie (BEO/22 0310814).
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Received 15 March 1999;
revised 28 May 1999;
accepted 14 June 1999.