Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany1
Area Microbiologia, Dpto Ecologia, Genetica y Microbiologia, Facultad de Biologia, Universidad de Leon, Campus de Vegazana, E-24071 Leon, Spain2
Author for correspondence: Jörn Kalinowski. Tel: +49 521 106 4825. Fax: +49 521 106 5626. e-mail: joern.kalinowski{at}genetik.uni-bielefeld.de
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ABSTRACT |
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Keywords: stringent response, (p)ppGpp, Corynebacterium, relA, spoT
The GenBank accession number for the 5·25 kb rplK region reported in this paper is AF130462.
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INTRODUCTION |
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There are two different sources of (p)ppGpp accumulation in Escherichia coli, depending on the nature of the nutritional stress imposed. The RelA protein, responsible for the (p)ppGpp biosynthesis during amino acid deprivation, is a ribosome-associated enzyme catalysing the transfer of a pyrophosphoryl group from ATP to the 3'-hydroxyl position of GTP or GDP. The enzyme is active when uncharged codon-specific tRNAs bind to the ribosomal A-site (Cochran & Byrne, 1974 ; Goldman & Jakubowski, 1990
). Furthermore, a functional 50S ribosomal protein L11, encoded by the rplK gene, is required for the activation of the RelA protein and thus for the synthesis of (p)ppGpp (Friesen et al., 1974
). However, relA mutant strains continue to accumulate (p)ppGpp in a spoT gene-dependent manner during energy-source exhaustion (Ryals et al., 1982
; Murray & Bremer, 1996
). The absence of all detectable (p)ppGpp in relA/spoT double mutants (Xiao et al., 1991
) and the fact that RelA and SpoT have extensive amino acid sequence similarity throughout their length (Metzger et al., 1989
) led to the proposal that SpoT is a bifunctional enzyme, responsible for (p)ppGpp degradation and for the second source of (p)ppGpp-forming activity in E. coli (Murray & Bremer, 1996
).
In contrast to the situation described for E. coli, data from genome sequencing projects of Mycobacterium tuberculosis (Cole et al., 1998 ), Bacillus subtilis (Kunst et al., 1997
), and Mycoplasma genitalium (Fraser et al., 1995
) revealed the presence of only one relA/spoT homologue in each of these bacteria. Moreover, the relA/spoT homologous genes from Streptococcus equisimilis H46A and Streptomyces coelicolor A3(2) are bifunctional, encoding a (p)ppGpp synthetase and a (p)ppGpp degrading activity (Mechold et al., 1996
; Martinez-Costa et al., 1998
). These findings supported the idea that in some bacteria only one gene might substitute for both functions encoded by the relA and spoT genes of E. coli. However, in B. subtilis and S. coelicolor A3(2) it has been shown that, like in E. coli, a functional rplK gene product is required for (p)ppGpp accumulation upon amino acid starvation (Smith et al., 1980
; Ochi et al., 1997
).
The Gram-positive soil bacterium Corynebacterium glutamicum is the most important micro-organism involved in the industrial production of amino acids such as L-glutamate, L-lysine and L-isoleucine (Malumbres & Martin, 1996 ). In the past decade, molecular genetic techniques have been developed for this micro-organism and numerous genes involved in amino acid biosynthesis have been cloned and characterized. Since then, the industrial production of selected amino acids has been improved by genetic engineering and amplification of relevant structural genes. More recently, a relA/spoT homologous gene from C. glutamicum, termed rel, has been reported and shown to encode a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp degrading activities (Wehmeier et al., 1998
). Moreover, RNA synthesis and growth rate of Brevibacterium flavum are inversely correlated with the cellular ppGpp concentration (Ruklisha et al., 1995
), supporting an essential role of the rel gene in the stringent response of this bacterium.
In the present study, we continue our work on the (p)ppGpp metabolism of C. glutamicum with the identification and molecular characterization of the rplK gene showing significant similarity to ribosomal large-subunit proteins L11. (p)ppGpp accumulation assays in wild-type and in rplK mutant strains strongly indicates that the rplK gene is indispensable for (p)ppGpp accumulation following amino acid deprivation in C. glutamicum.
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METHODS |
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Polymerase chain reactions.
The synthetic oligonucleotides P1-up (5'-AGGAGCAGGCTGTTGTCACC-3'), P1-down (5'-extension-CGCCGTGAGC-5'-GCCAACTGGAGGAGCAGGGT-3'), and P2-up (5'-extension-TCCAGTTGGC-5'-GCTCACGGCGTCAACATCAG-3'), P2-down (5'-GCGGATAGCTACGTCGATGG-3') were used for the amplification of two 600 bp fragments comprising the 5' and 3' regions of the C. glutamicum rplK gene, respectively, with Pfu DNA polymerase (Stratagene). The construction of the chromosomal 12 bp deletion within the rplK gene was analysed with the primers Pdel-up (5'-TGGCCCAGCACTTGGT-3') and Pdel-down (5'-GCAATAGCAACGTCGA-3'). The oligonucleotides P1-up and P2-down were used to amplify a fragment comprising the rplK wild-type gene. PCR experiments were carried out with a PCT-100 thermocycler (MJ Research). Initial denaturation was conducted at 94 °C for 2 min followed by 90 s denaturation, 90 s annealing at 58 °C and extension at 72 °C for 2 min. This cycle was repeated 30 times followed by a final extension step for 10 min at 72 °C. PCR products were purified using the PCR Purification Spin Kit (Qiagen).
Southern hybridization.
Chromosomal DNA of C. glutamicum was digested with restriction enzymes, separated on 0·8% agarose gels, and blotted onto Hybond-N nylon membranes (Amersham) using the vacuum blotter Vacu-Gene (Pharmacia). Fixation of DNA, labelling of DNA probes and hybridization were performed with the non-radioactive DIG DNA Labelling and Detection Kit from Boehringer.
DNA sequencing and sequence analysis.
During analysis of transposon insertions in C. glutamicum, the Tn5516invers insertion of the transconjugant C. glutamicum KM6 was cloned by applying the plasmid rescue technique (Tauch, 1996 ). For this purpose, total DNA of C. glutamicum KM6 was isolated, cut with SalI and religated. The ligation mixture was subsequently transformed into E. coli DH5
MCR. The resulting plasmid pKM6-6, carrying a 5·8 kb contiguous DNA fragment of the C. glutamicum chromosome, was used as a template for nucleotide sequencing by primer walking, performing the recommended procedures of the Prism Ready Reaction Dye Deoxy Termination Kit (Applied Biosystems) in conjunction with a Prism 377 DNA sequencer (Applied Biosystems). Computer-assisted compilation of the DNA sequences was conducted with the Staden software package (Staden, 1986
). Searches for amino acid similarities were carried out with the BLAST programs (Altschul et al., 1997
). Global alignments between protein sequences were calculated with the ALIGN computer program (Myers & Miller, 1988
). The prediction of protein secondary structures was calculated by the neural network system PredictProtein (Rost et al., 1995
).
Screening for spontaneous and induced thiostrepton-resistant C. glutamicum mutants.
For obtaining spontaneous thiostrepton-resistant colonies, different amounts of an exponentially growing culture (1061012 cells) were spread on LB agar containing 1, 2·5, 5, or 10 µg thiostrepton ml-1, respectively, and were incubated for 10 d at 30 °C. The screening for induced thiostrepton-resistant mutants was performed by chemical mutagenesis using ethyl methanesulfonate (EMS). An exponentially growing culture was concentrated 10-fold by centrifugation and resuspended in 0·1 M sodium phosphate buffer (pH 7·0) containing 1 mg EMS ml-1 and incubated at 30 °C for 1 h. Cells were then washed twice with LB medium and plated directly onto LB agar supplemented with different thiostrepton quantities (see above) and incubated at 30 °C.
Construction of a plasmid-encoded rplK deletion derivative.
A 1·2 kb fragment comprising a 12 bp deletion derivative of the rplK gene was amplified from the C. glutamicum chromosome using PCR with the oligonucleotides P1-up, P1-down, P2-up and P2-down (illustrated in Fig. 3). The PCR primers were deduced from the known nucleotide sequence (P1-up, nt 32913310; P1-down, nt 39163897; P2-up nt 39293948; P2-down, nt 45344515) in such a way that P1-down and P2-up contained 10 bp 5' extensions which matched exactly the 5' end of P2-up (nt 39293938) and P1-down (nt 39163907), respectively. First, two separate 600 bp fragments were amplified from the target sequence. The first product, termed rplk-part1, was obtained with P1-up and P1-down and comprised the 5' part of the rplK gene (nt 187) together with a 10 bp extension corresponding to nt 100109 of rplK, thereby carrying a 12 bp gap within the amplified rplK portion compared to the used chromosomal template. The second fragment, named rplk-part2, was obtained with P2-up and P2-down and consisted of the rplK 3' region (nt 78435) carrying the same 12 bp gap ranging from nt 8899 within the amplified rplK part. Accordingly, these two rplK PCR products shared a 20 bp overlap region. When these intermediate products were mixed together, melted and reannealed, the top strand of rplk-part1 could anneal to the bottom strand of rplk-part2 in such a way that the two strands acted as primers on one another. Extension of this overlap and addition of the primers P1-up and P2-down created a 1·2 kb fragment which carried a 12 bp deletion derivative of the rplK gene. The purified 1·2 kb PCR product was subsequently cloned into the SmaI-digested E. coli vector pK18mobsacB, yielding p
RPLK. Therefore, p
RPLK comprised a mutant rplK gene, in which the 12 bp deletion resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the corresponding L11 protein.
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Transcripitional assay.
RNA of C. glutamicum was isolated by means of the Ribolyser instrument and the Hybaid Ribolyser RNA isolation kit. Blotting and hybridization experiments were performed as described by Sambrook et al. (1989) . Hybridization signals were detected with the chemoluminescence substrate CSPD (Roche) and quantified densitometrically using the LabImage computer program (Labsoft). Gene probes of rplK and rplA were generated by PCR amplification using the Pwo DNA polymerase and the oligonucleotides rplK1 (nt 37583777), rplK2 (nt 42734292), rplA1 (nt 44904509) and rplA2 (nt 49764995), respectively. Subsequently, the PCR products were cloned into the vector pZEro-2 (Invitrogen). In vitro labelling of the cloned DNA fragments was performed with the DIG DNA labelling kit (SP6/T7) from Roche.
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RESULTS |
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A DNA fragment comprising the rplK deletion derivative was obtained by a PCR strategy and was cloned into pK18mobsacB, yielding pRPLK. A detailed experimental procedure of this amplification is outlined in Methods and in Fig. 3
. Subsequently, p
RPLK was transferred to E. coli S17-1 and integrated into the chromosome of C. glutamicum RES167 by conjugation resulting in C. glutamicum LW45. Excision of the plasmid can be selected for by growing the cells on LB agar containing 10% sucrose. Cells able to grow on this medium have lost the plasmid due to a second crossover event that either restores the wild-type gene arrangement or leads to a mutant strain carrying a defined 12 bp deletion in the chromosome. However, all 97 clones tested contained the wild-type gene arrangement.
In a further approach, a DNA fragment comprising the wild-type rplK gene was obtained by PCR and was cloned into the SmaI-digested E. coliC. glutamicum shuttle vector pECM3. The resulting plasmid, pRPLK, was subsequently transferred to C. glutamicum LW45 by conjugation. With the wild-type rplK copy provided in trans, we were able to select a second crossover and to replace the chromosomal rplK gene which was verified by sequencing of a chromosomal rplK PCR fragment originating from a sucrose-resistant clone (data not shown). The elimination of the plasmid-encoded rplK wild-type copy from the cell was subsequently achieved by heat-curing of pRPLK (Schäfer et al., 1994a ). The complete loss of pRPLK was verified by Southern blot experiments using labelled pRPLK as a probe (data not shown). Finally, the resulting strain C. glutamiucm RES167
rplK carried a chromosomal 12 bp deletion within the rplK gene corresponding to the tetrapeptide Pro-Ala-Leu-Gly from the L11 protein.
The deletion within the rplK gene leads to a tolerance of C. glutamicum against thiostrepton
The RplK protein of E. coli forms part of the target site for the antibiotic thiostrepton, which binds the same domain of the 23S rRNA (Ryan et al., 1991 ). Therefore, mutations in the RplK protein render ribosomes thiostrepton resistant (Friesen et al., 1974
). Since our former attempts to isolate rplK mutants of C. glutamicum by screening for spontaneous or induced thiostrepton-resistant colonies failed, we have analysed whether the constructed deletion within the rplK gene confers thiostrepton resistance in C. glutamicum. The growth of C. glutamicum RES167 was inhibited in the presence of 60 ng ml-1 thiostrepton in liquid growth medium expressed as the 50% value of the OD580, whereas the rplK mutant strain RES167
rplK grew in the presence of thiostrepton concentrations of up to 150 ng ml-1. Thus, the C. glutamicum rplK mutant exhibited a 2·5-fold increased tolerance of thiostrepton.
The C. glutamicum rplK mutant is significantly impaired in (p)ppGpp accumulation upon amino acid starvation
To assess the effect of the mutant rplK gene on (p)ppGpp accumulation, C. glutamicum RES167 rplK was subjected to amino acid starvation. Conditions of amino acid deprivation were induced by supplementation with 1 mg DL-serine hydroxamate ml-1, which inhibits tRNASer aminoacylation, and with 0·5 mg L-valine ml-1, which resulted in isoleucine starvation (Cashel et al., 1996
). Under such stress conditions, we detected (p)ppGpp in nucleotide extracts of C. glutamicum RES167 which co-migrates with (p)ppGpp synthesized by the E. coli wild-type (Fig. 4
, lanes 1 and 3). This starvation response was completely abolished in E. coli CF1652 (
relA) and C. glutamicum RES167
rel, a mutant lacking a functional (p)ppGpp synthetase (Wehmeier et al., 1998
) (Fig. 4
, lanes 2 and 4). The C. glutamicum rplK mutant is significantly impaired in its ability to form (p)ppGpp following nutritional shift-down (Fig. 4
, lane 5). A computer-assisted analysis of the spot intensities, using the WinCam program, revealed only 14±5% (p)ppGpp compared to the (p)ppGpp level detected in the C. glutamicum wild-type. The (p)ppGpp accumulation assignment was verified by comparing the nucleotide activities of a limited series of sample dilutions (data not shown). The ability of C. glutamicum RES167
rplK to produce (p)ppGpp could be completely restored by introducing a plasmid-encoded rplK wild-type allele into the mutant strain (Fig. 4
, lane 6), indicating that the rplK gene is required for (p)ppGpp accumulation upon amino acid deprivation.
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DISCUSSION |
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E. coli rplK mutants are strongly impaired in their ability to accumulate (p)ppGpp following a nutritional shift-down and exhibit, like relA mutants, a relaxed control of RNA synthesis. Therefore, the rplK locus was originally termed relC (Cashel et al., 1996 ). Almost identical results have been obtained in several other bacteria including B. subtilis (Smith et al., 1980
), Bacillus megaterium (Stark & Cundliffe, 1979
), S. coelicolor A3(2) (Ochi et al., 1997
), and S. griseus (Kawamoto et al., 1997
), where mutations within the respective rplK/relC gene lead to an abolished stringent response. To determine the role of the C. glutamicum rplK gene in (p)ppGpp accumulation, we constructed and analysed an rplK mutant strain. All rplK mutants described so far have been isolated either by chemical mutagenesis or due to their property to confer thiostrepton resistance (Ochi, 1990
). In contrast to these observations, thiostrepton-resistant C. glutamicum colonies could not be obtained under a variety of different screening procedures. Moreover, attempts to disrupt the rplK coding region, or to delete larger internal parts of rplK using the sacB system, which facilitates the detection of an allelic exchange by homologous recombination (Schäfer et al., 1994b
) were unsuccessful, providing evidence that the RplK protein might be essential in C. glutamicum. Finally, the strain C. glutamicum RES167
rplK was constructed which carries a 12 bp in-frame deletion within the rplK gene, resulting in the loss of the tetrapeptide Pro-Ala-Leu-Gly from the L11 protein. The deleted sequence represents the most highly conserved region of protein L11 in Gram-positive and Gram-negative bacteria and was shown to nearly prevent (p)ppGpp accumulation under amino acid starvation conditions in S. griseus (Kawamoto et al., 1997
) and S. coelicolor A3(2) (Ochi et al., 1997
).
(p)ppGpp accumulation assays in C. glutamicum wild-type and the rplK mutant strain established that the rplK gene is required for (p)ppGpp accumulation. Synthesis of (p)ppGpp invoked by amino acid starvation was almost abolished in the rplK mutants but not in the wild-type strain. The residual (p)ppGpp level corresponding to approximately 14±5% in the rplK mutant compared with the C. glutamicum wild-type agrees well with the (p)ppGpp accumulation level of about 10% remaining in rplK mutant strains of E. coli (Friesen et al., 1974 ), S. griseus (Kawamoto et al., 1997
) and S. coelicolor A3(2) (Ochi et al., 1997
). The deletion within the C. glutamicum rplK gene leads only to a 2·5-fold increase in the tolerance against thiostrepton, which is much lower than the 14100-fold resistance known from the rplK mutant strains of S. griseus and S. coelicolor A3(2), respectively. However, the C. glutamicum wild-type is about 1327-fold more sensitive to thiostrepton than both Streptomyces parental strains. Western blotting analysis revealed that the ribosomes from the S. griseus and S. coelicolor A3(2) rplK mutants incorporate the corresponding mutant L11 protein normally, although the level of incorporation is only approximately one-third to one-eighth of that observed with the wild-type L11 protein (Kawamoto et al., 1997
; Ochi et al., 1997
). Since C. glutamicum RES167
rplK, like the S. griseus rplK mutant, lacks the highly conserved internal 12 bp region, it seems likely that ribosomes from the C. glutamicum mutant strain also contain apparently lower amounts of mutant L11 protein, resulting in the strong impairment in (p)ppGpp accumulation. Despite the result of complementation in trans of the rplK mutant with the wild-type gene, we could not finally exclude the possibility that the defined 12 bp in-frame deletion within rplK may cause a polar effect on rplA which itself may eventually contribute to the defect in (p)ppGpp accumulation. Northern analysis revealed that the rplK and rplA genes of C. glutamicum form a bicistronic operon since a transcript covering both genes was detected. However, the same transcription patterns with slightly higher levels of the rplKrplA transcript were observed in the defined rplK mutant strain, suggesting that effects on the stability of the messenger can be neglected.
Recently, we isolated a relA/spoT homologous gene from C. glutamicum, designated rel, which probably encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGppase activities. A rel mutant strain failed to synthesize (p)ppGpp in response to amino acid starvation (Wehmeier et al., 1998 ). In the present study, we established that a functional 50S ribosomal protein L11 in C. glutamicum is indispensable for (p)ppGpp accumulation upon nutritional deprivation. Therefore, it appears possible that the C. glutamicum Rel protein, like the homologues of E. coli (Cochran & Byrne, 1974
) and S. coelicolor A3(2) (Martinez-Costa et al., 1998
), is ribosome-associated, requiring the RplK protein for activation of its (p)ppGpp synthetase function. With the molecular characterization of the rel and rplK genes from C. glutamicum we have identified two genes involved in (p)ppGpp synthesis in this industrially important micro-organism. Both genes are therefore involved in a global regulatory network response which might also influence amino acid production. This aspect is under current investigation in our laboratory.
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ACKNOWLEDGEMENTS |
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Received 26 October 2000;
accepted 20 November 2000.