National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan1
Author for correspondence: Kozo Ochi. Tel: +81 298 38 8125. Fax: +81 298 38 7996. e-mail: kochi{at}affrc.go.jp
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ABSTRACT |
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Keywords: RNA polymerase, stringent response
Abbreviations: Act, actinorhodin; Red, undecylprodigiosin; Rif, rifampicin
a Present address: Department of Pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110015, China.
b Present address: Mitsubishi Kagaku Institute of Life Sciences, Yokohama Research Center, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan.
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INTRODUCTION |
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In a previous study (Hu et al., 2002 ), we reported that the introduction of certain Rif-resistance mutations (rif) into S. lividans can activate Act and Red production in this organism. These rif mutations were frequently found in the so-called rif domain within the rpoB gene, which encodes the RNA polymerase ß-subunit. In the present study, we have attempted to characterize these rif mutations by using both genetic and physiological approaches, including proteome analysis.
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METHODS |
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Isolation and manipulation of DNA.
Plasmid and total DNA were isolated from S. lividans as described by Kieser et al. (2000) . Protoplast transformation was also done as described by Kieser et al. (2000)
. Southern analysis was performed using digoxigenin (DIG)-labelled probes made by random oligonucleotide priming (DIG DNA-labelling kit; Boehringer Mannheim). E. coli strains were grown and transformed using standard protocols (Sambrook et al., 1989
).
Mutation analysis of rpoB.
All primers used for checking chromosomal mutations in rpoB were designed using sequence information obtained from S. coelicolor M145 (accession no. AL160431; Redenbach et al., 1996 ). The nucleotide sequence for the PCR fragment was determined by the dideoxynucleotide chain termination method using the BigDye Terminator Cycle Sequencing Kit (Perkin Elmer).
Gene-replacement analysis of rpoB.
The procedure used for gene-replacement analysis is illustrated in Fig. 4. Total chromosomal DNA was prepared from the two rif mutants KO-417 (rif-17 Act-positive) and KO-418 (rif-17 rif-18 Act-positive). A 4354 bp SacISacI fragment from KO-417 and KO-418 was cloned into the SacI site of the multiple-cloning sites in a pBluescriptSK(+) vector to generate pLC1 and pLC2, respectively. The insert includes the rpoB coding region but lacks 15 aa residues at the carboxyl terminus. A 1·1 kb EcoRIEcoRI fragment containing the apramycin-resistance gene (aac(3)IV; accession no. X99319) was cloned into both pLC1 and pLC2 in a step-wise manner, generating pLC3 and pLC4, respectively. Plasmids pLC3 and PLC4 were passaged through the methylation-deficient E. coli strain DM1 (dam dcm) and introduced into S. lividans strains as described previously (Kieser et al., 2000 ). The R2YE plates were flooded with 1 ml of an apramycin (Sigma) solution, to give a final concentration of 50 µg ml-1. Integration and looping-out of the plasmids by homologous recombination were confirmed by Southern hybridization.
Western blotting and RT-PCR analysis.
Western analysis was carried out as described previously (Hu et al., 2002 ). RT-PCR was carried out by using the Thermoscript RT-PCR System Kit (Invitrogen).
Assay for ppGpp and determination of RNA synthesis.
The intracellular ppGpp content was assayed as described by Ochi (1987) using HPLC analysis. RNA synthesis after Casamino acid deprivation or during growth in a Casamino acid medium was determined by measuring [2-14C]uracil incorporation into acid-precipitable material, as described previously (Ochi, 1990a
).
Two-dimensional gel electrophoresis.
Cells were collected from a GYM plate covered with a cellophane sheet. They were then disrupted by sonication three times for 30 s on ice, and centrifuged at 14000 g for 20 min. The supernatants were used as protein extracts and 150 µg total protein from each sample was applied to an Immobiline Dry Strip (pH 47, 18 cm; Amersham Pharmacia) for isoelectric focusing using the Multiphor II Electrophoresis Unit (Amersham Pharmacia). An ExcelGel (XL SDS 1214%; Amersham Pharmacia) was used for the second dimension SDS-PAGE.
Peptide-mass-fingerprinting analysis and N-terminal-sequencing analysis.
The gels were stained with Coomassie blue. Spots of interest were cut out and subjected to peptide-mass-fingerprinting analysis and N-terminal-sequencing analysis. Gel pieces were washed and dried under vacuum, before being digested with trypsin (Promega). After trypsin treatment, peptides were extracted with 25 µl of 50% acetonitrile/5% trifluoroacetic acid (TFA). The extracts were dried by using a Speed-Vac and reconstituted by adding 6 µl of 50% acetonitrile/0·1% TFA. Resulting samples were spotted onto a MALDI-TOF/MS sample target with -cyano-4-hydroxycinnamic acid (Fluka). Angiotensin II (human; Sigma) and insulin chain B (bovine; Sigma) were used for external calibration. Samples were analysed using a REFLEX II MALDI mass spectrometer (Bruker). Mascot (Matrix Science) was used to identify the protein from the mass data. For N-terminal-sequencing analysis, gel spots from the two-dimensional polyacrylamide gels were blotted onto a PVDF membrane. After staining, the spots were cut out and the membrane was subjected directly to N-terminal-sequencing analysis using a G1000A Protein Sequencer (Hewlett Packard).
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RESULTS |
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Strikingly, among the rif mutants that were generated spontaneously from the relC strain KO-421, the Act-positive phenotype was detected at a frequency as high as 80% (81 out of 101). Two representative mutants, KO-422 (relC rif-1) and KO-423 (relC rif-2), can be found in Table 1. Both mutants possess a point mutation within rpoB, as detected by DNA sequencing (Table 2
). Act production by the relC rif double mutant KO-422 is shown in Fig. 5
, as an example. These results, together with the results from the ppGpp assay, indicate that the rif effect on antibiotic production activation can be provoked even in the relC genetic background.
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Effect of Casamino acid deprivation on RNA synthesis
The rel mutants, including relA and relC, are characterized by the relaxed response (i.e. failure to inhibit stable RNA synthesis) to amino-acid limitation (Ochi, 1990a , b
). We hypothesized that the rif mutations that conferred the Act-positive phenotype may behave like stringent RNA polymerases during growth in nutritionally rich media. To assess this possibility, we analysed the rate of RNA synthesis during growth in a nutritionally rich medium (i.e. synthetic medium supplemented with 2% Casamino acids), using the rif mutants KO-417 and KO-418 (Fig. 6a
). These strains were grown to mid-exponential phase [100 mg dry cell wt (100 ml culture)-1], and then [2-14C]uracil was added to the culture, followed by a further 60 min incubation. Strikingly, rif mutant KO-417 revealed a fourfold reduction in RNA synthesis, when compared to the wild-type strain. The rif double mutant KO-418 exhibited a less-pronounced reduction in RNA synthesis, but the reduction was still significant (Fig. 6a
) despite having no discernible effect on growth (see Fig. 2
). Likewise, the relC rif double mutant KO-422 exhibited significantly reduced RNA synthesis compared to the parental relC strain KO-422 (Fig. 6b
), although the mutant also showed the relaxed response upon nutritional shift-down (Fig. 3b
), reflecting the inability of the mutant to accumulate normal levels of ppGpp (Fig. 3a
). Thus, the rif mutants exhibiting the Act-positive phenotype have a RNA polymerase with reduced activity for RNA synthesis during their growth in a nutritionally rich medium.
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DISCUSSION |
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The present study provides evidence for the significance of the growth rate of S. lividans in antibiotic production; the growth rate of the organism was closely linked to the rate of RNA synthesis, as seen in the order wild-type strain > double rif mutant (KO-418) > single rif mutant (KO-417). It is therefore likely that both the timing and the extent of antibiotic production by Streptomyces spp. are crucially decided by the physiological status of the RNA polymerase within the cell. ppGpp, a mediator for the stringent response, has been demonstrated to directly bind to the RNA polymerase ß-subunit in E. coli (Chatterji et al., 1998 ). Reddy et al. (1995)
also provided evidence for the location of the ppGpp-binding site on the E. coli RNA polymerase and the proximity relationship with the rif-binding domain. Detailed mapping of the promoter recognition domain on the ß-subunit has been done using a collection of various RNA polymerase ß-subunit mutants, each with a single amino-acid substitution. Thus, the domains for Rif sensitivity, ppGpp sensitivity, promoter selectivity and
assembly were found to be lined up along the rpoB gene, which encodes the RNA polymerase ß-subunit (Ishihama, 1988
). From the result obtained by X-ray analysis of the core RNA polymerase (Zhang et al., 1999
), Toulokhonov et al. (2001)
proposed that the binding of ppGpp is allosteric and that the binding site is modular. Previous reports, in which various bacteria have been studied, have demonstrated that mutations in rpoB are responsible for the acquisition of resistance to Rif (Aboshkiwa et al., 1995
; Jin & Gross, 1988
; Singer et al., 1993
). Results recently obtained in E. coli show that RNA polymerase mutants selected to confer prototrophy to a
relA
spoT strain can mimic the effect of ppGpp on the wild-type RNA polymerase (Barker et al., 2001
). Therefore, it is reasonable to consider that the RNA polymerase with a rif-type ß-subunit may be structurally similar to an RNA polymerase that has been modified by ppGpp, because numerous genetic analyses revealed that rif mutations frequently circumvent the ppGpp0 phenotype. Indeed, as demonstrated in E. coli, the mutant RNA polymerase may have altered promoter selectivity (Ishihama et al., 1990
). In particular, the ppGpp-independent stringent RNA polymerases have been described and the model for linking the dual aspects of the stringent response has been proposed (Zhou & Jin, 1998
). It is conceivable that the altered conformational status of the RNA polymerase resulting from rif-17 in S. lividans gave rise to different promoter selectivity (or affinity), directly or indirectly leading to the increased actII-ORF4 expression. Although the RNA polymerase with the rif-17 mutation behaved like a stringent RNA polymerase with respect to RNA synthesis (Fig. 6
), we can not rule out the possibility that the mutant RNA polymerase generated different promoter selectivity that was capable of activating different pathways for the activation of antibiotic biosynthesis and, hence, did not behave as a stringent RNA polymerase. For instance, the clear difference in certain gene expression (see below) can be originated by a stringent RNA polymerase or simply by a modified promoter selectivity of the mutant RNA polymerase. Although the rif-17 mutation resulted in the abrogation of growth, which was apparently due to severe suppression of RNA synthesis (Fig. 6a
), rif-18 (just adjacent to rif-17) could almost completely restore growth (Fig. 2
). The effect of rif-18 on the ß-subunit can also be explained by the subsequent alteration of the three-dimensional structure of this subunit.
The increase in the production of glutamine synthetase (type II) and oxidoreductase by introducing a rif mutation (Fig. 7) into S. lividans was dramatic. Glutamine synthetase, responsible for the synthesis of glutamine from
and glutamate, is a key enzyme in
assimilation and is regulated by nitrogen availability in micro-organisms, including Streptomyces strains (Fisher, 1999
). At least two types of glutamine synthetase exist in bacteria, GSI (encoded by glnA) and GSII (encoded by glnII). Enteric bacteria and Bacillus subtilis only possess the GSI type, but Streptomyces strains are known to possess the eukaryotic-type glutamine synthetase GSII as well as GSI (Weisschuh et al., 2000
). The role of the GSII enzyme in nitrogen metabolism in Streptomyces spp. is unclear. In nitrogen-fixing bacteria, GSII is preferentially expressed during nitrogen-limited growth and nitrogen fixation (Fisher, 1992
). There are many kinds of oxidoreductases in S. coelicolor, as assigned by the S. coelicolor genome-sequencing project (http://www.sanger.ac.uk/Projects/S_coelicolor). The oxidoreductase (GenBank no. CAC37883) that was highlighted in this study is encoded by the SC1G7.08c gene, which is located near the type I polyketide synthesis gene cluster. Although our results implicate the intrinsic role of glutamine synthetase and oxidoreductase in secondary metabolism in S. lividans, further investigations are required to establish a causal relationship between these two enzymes and secondary metabolism.
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ACKNOWLEDGEMENTS |
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Received 30 April 2002;
revised 10 July 2002;
accepted 20 August 2002.