National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
Correspondence
Kozo Ochi
kochi{at}affrc.go.jp
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ABSTRACT |
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INTRODUCTION |
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Antibiotic production in Streptomyces spp. is believed to occur in a growth-phase-dependent manner; the initiation of antibiotic biosynthesis, including that of Act and Red, usually starts at the transition between vegetative growth and morphological development (Strauch et al., 1991). The biosynthetic pathways for Act and Red are positively regulated at the transcriptional level by their own pathway-specific regulatory proteins, ActII-ORF4 and RedD (Strauch et al., 1991
; Takano et al., 1992
), the expression of which occurs at the end of exponential phase. A recent report by Lai et al. (2002)
on various rif mutants provided evidence for the significance of the growth rate in antibiotic production by S. lividans. The growth rate of the organism was closely linked to the rate of RNA synthesis, suggesting 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. This proposal was supported by the fact that antibiotic production by both S. coelicolor and S. lividans is dramatically enhanced or activated by introducing certain mutations into the rpoB gene (encoding the RNA polymerase
-subunit) in the genetic backgrounds of relA and relC (Hu et al., 2002
; Xu et al., 2002
). Apart from the regulation at the transcription level, we have recently reported that certain mutations in rpsL (encoding the ribosomal protein S12) activate antibiotic production in various bacteria (Hesketh & Ochi, 1997
; Hosoya et al., 1998
; Okamoto-Hosoya et al., 2000
, 2003
; Shima et al., 1996
). The substitution of Lys-88 with Glu (K88E) in the S12 protein could elicit the ability to produce Act and Red in S. lividans 66, and enhanced the production of those antibiotics in S. coelicolor A3(2) more than fivefold. This mutation conferred a high level of resistance to streptomycin in addition to activation of antibiotic production. On the other hand, the substitution of Lys-43 with Asp (K43D) did not affect antibiotic production, although this mutation also conferred a high level of resistance to streptomycin. The transcription and translation levels of pathway-specific regulators (ActII-ORF4 and RedD) were both significantly increased in the K88E mutant, as demonstrated by RNase protection assay (S. Okamoto & K. Ochi, unpublished results) and by Western blot analysis (Hu & Ochi, 2001
). However, the mechanism by which ribosomal mutation activates antibiotic production has remained obscure.
The ribosomal protein S12 is a component of the 30S ribosomal subunit in bacteria. The S12 protein is best characterized with respect to a role in the recognition of cognate tRNA for maintaining accurate translation (for a review, see Kurland et al., 1990). Resistance to streptomycin is often conferred by mutations in the rpsL gene. Of the 124 amino acid residues that compose the Escherichia coli S12 protein, about 20 kinds of substitutions are found to confer either streptomycin resistance (SmR) or streptomycin dependence (SmD) (Timms et al., 1992
; Toivonen et al., 1999
). Most ribosomes from SmR mutants display an increased translational accuracy (restrictive phenotype), except for the K43R mutation, which shows a reduced translational accuracy (non-restrictive phenotype) (Carter et al., 2000
; Inaoka et al., 2001
). Using various such restrictive mutations (mainly those that occurred at the position Lys-43), a number of studies have demonstrated that the mutant ribosome has a decreased binding affinity both of the ternary complex (aminoacyl-tRNA-GTP-EF-Tu) for the ribosome (Bilgin et al., 1992
), and of the A-site for aminoacyl-tRNA (Karimi & Ehrenberg, 1996
). The mutation K88E which we found in Streptomyces has never been isolated to date from E. coli (Gregory et al., 2001
). In the present study, we characterized the K88E mutant cells, with emphasis on the novel property of mutant ribosomes. This report is believed to be the first to offer a rationale for the observed antibiotic overproduction caused by rpsL mutations.
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METHODS |
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[3H]Leucine incorporation.
Bacterial strains were grown to mid-exponential phase in YEME medium, then 0·2 µCi (7·4 kBq) [3H]leucine (200 µM) was added to the 10 ml culture and further incubated. Aliquots (1 ml) of the mixture were taken at 0, 5, 10, 30 and 60 min after the addition of leucine, mixed with 1 ml 10 % trichloroacetic acid, and kept on ice for 30 min to precipitate the protein from solution. The precipitated protein was collected by filtration on nitrocellulose filter (pore size 0·45 µm) and washed with 10 ml 5 % TCA. The filters (containing protein) were dried and their radioactivity was measured by liquid scintillation counting.
Determination of RNA and protein synthesis after amino acid depletion.
Bacteria were grown in CD medium supplemented with 3 % Casamino acids. Cultures at mid-exponential growth phase (3 mg dry cell weight ml-1) were divided into two portions, filtered, washed and resuspended in fresh CD medium with or without Casamino acids, which contained [14C]uracil (0·2 µCi, 7·4 kBq, 100 µM) and [3H]leucine (2 µCi, 74 kBq, 200 µM). The rates of total RNA and protein synthesis were determined by measuring the incorporation of radioactivity into the acid-insoluble portion.
Poly(U)-directed cell-free translation.
We used the poly(U)-directed cell-free translation system of Legault-Demare & Chambliss (1974) with some modifications. Cells of S. coelicolor strains grown to mid-exponential phase in YEME medium were harvested by filtration, and washed with standard buffer (10 %, v/v, glycerol, 10 mM magnesium acetate, 30 mM ammonium acetate, 6 mM 2-mercaptoethanol, 10 mM Tris/HCl, pH 7·7) containing 1 mM PMSF. The washed cells were ground with abrasive aluminium oxide powder (Wako), and ultracentrifuged (150 000 g, 4·5 h) to separate the ribosome (pellet) and supernatant (designated S-150) fractions. The ribosome fraction was further washed once with the standard buffer. Both the ribosome and S-150 fractions were dialysed against 60-fold volume of standard buffer for 6 h, divided into small aliquots, frozen immediately in liquid nitrogen, and stored at -80 °C until used for experiments. Ribosome mixtures containing 5 A260 units of ribosome ml-1, 1·0 mg protein of S-150 ml-1, 55 mM HEPES/KOH (pH 7·5), 1 mM dithiothreitol, 210 mM potassium acetate, 27·5 mM ammonium acetate, 10·7 mM magnesium acetate and 68 mM folinic acid were incubated at 30 °C for 10 min to remove the endogenous mRNA. The reaction mixture (100 µl) contained 60 µl ribosome mixture, 0·12 µmol ATP, 0·08 µmol GTP, 0·064 µmol 3',5'-cAMP, 8 µmol creatine phosphate, 0·025 mg creatine kinase, 0·05 units RNase inhibitor (recominant solution; Wako), 15 µg E. coli tRNA, 0·04 µmol each of 20 L-amino acids (but lacking phenylalanine or leucine), and 22 pmol [14C]phenylalanine (0·01 µCi, 0·37 kBq) or [3H]leucine (0·94 µCi, 34·8 kBq). The reaction was initiated by the addition of 75 µg poly(U) and the mixtures were incubated at 30 °C for the appropriate time; 1 ml 10 % trichloroacetic acid was subsequently added (to stop the reaction) and the mixtures were boiled for 15 min. Precipitated proteins were collected on nitrocellulose filters, and the incorporation of [14C]phenylalanine or [3H]leucine into the acid-insoluble fraction was determined by liquid scintillation counting. The missense error frequency was expressed as the ratio of incorporation of [3H]leucine to incorporation of [14C]phenylalanine (Inaoka et al., 2001
).
Polysome profiling.
Polysomes were prepared from cells using rapid-chilling methods as described by Godson & Sinsheimer (1967). Cultures were cooled within 10 s from 30 °C to 24 °C using an ice bath, and then frozen in liquid nitrogen. Frozen cells (0·2 g) were homogenized in 1 ml lysis buffer (15 mM MgCl2, 2 mM dithiothreitol, 10 mM Tris/HCl, pH 7·8) containing deoxyribonuclease (Wako) and protease inhibitor cocktail (Complete, Roche). The homogenates were centrifuged at 12 000 g for 5 min, then the supernatants were directly laid on a linear sucrose gradient (1545 %) in a gradient buffer (10 mM MgCl2, 100 mM NH4Cl and 2 mM dithiothreitol, 10 mM Tris/HCl, pH 7·8). The absorbance of the supernatant at 260 and 280 nm was used as an index of the concentration of the polysome preparation. The gradients were centrifuged at 180 000 g in a Beckman SW41 rotor at 4 °C for 3 h, and the distribution of polysomes on the gradients was recorded using BIOCOMP Piston Gradient Fractionator (Towa Kagaku) equipped with ATTO Bio-Mini UV Monitor (set at 254 nm).
Density-gradient sedimentation of the 70S complex.
Frozen cells were prepared and homogenized as described for polysome preparation (see above). The supernatants were centrifuged at 30 000 g for 30 min using a Beckman TLA 100.4 rotor at 4 °C (S-30 fraction). The supernatants were again centrifuged at 150 000 g for 1·5 h; the resulting pellets (P-150 fractions) were suspended in a basal buffer (100 mM NH4Cl, 2 mM dithiothreitol, 10 mM Tris/HCl, pH 7·8), and kept at 4 °C overnight. The suspensions were then centrifuged at 150 000 g for 6 h and the pellets resuspended in gradient buffers with appropriate concentrations of MgCl2. The samples were laid on a linear sucrose gradient (1040 %) in the same gradient buffer.
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RESULTS |
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K88E mutants show an aberrant growth phenotype
S. coelicolor and S. lividans both grow and disperse well when cultured in YEME medium. S. coelicolor wild-type strain 1147 grew rapidly in YEME medium with a temporary (2 h) cessation of growth at the mid-exponential growth phase (Fig. 1a), possibly due to depletion of the nitrogen source, as previously pointed out by Vohradsky et al. (2000)
. Later (at 42 h), cells entered into the stationary phase. On the other hand, strain KO-178 (K88E) showed a distinctive growth pattern: it grew rapidly until reaching the cessation point, but later grew slowly as if the cultures do not undergo stationary phase. Other rpsL mutants did not show this aberrant growth pattern as examined in 1258 (K43N) and KO-482 (K88R) strains (data not shown). The distinctive growth pattern was also detected in the S. lividans K88E mutant, TK24 (Fig. 1b
).
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K88E mutants sustain a high level of protein synthesis at the late growth phase
The incorporation of [3H]leucine into the protein fraction in living cells of both the parent and mutant strains was monitored as a measure of the level of protein synthesis. Strains were grown to various growth phases, and [3H]leucine was added to the culture, followed by a further 30 min incubation. In S. coelicolor wild-type strain 1147, protein synthesis was maximal at mid-exponential phase (29 h) and decreased as the cells aged to about half (Fig. 2a), indicating that protein synthesis activity decreased significantly after reaching the point of temporary growth cessation. In contrast, K88E mutant KO-178 sustained a high level of protein synthesis throughout the 60 h cultivation. This characteristic was more pronounced in S. lividans K88E mutant TK24, in which protein synthesis was enhanced, independent of the cell's age, although the activity at mid-exponential phase (20 h) was only one-third that of parental strain TK21 (Fig. 2b
). These results indicate that the K88E mutant cells are able to keep a high translation activity during the late growth phase. This conclusion was supported by the fact that K88E mutant cells displayed abundant polysomes (as indicated by arrows in Fig. 3b
) even at the late growth phase (4551 h), an indication of active protein synthesis. In the wild-type cells, the amount of polysomes decreased rapidly after 28 h (Fig. 3a
). Likewise, the K88E mutant cells (but not the wild-type cells) displayed a detectable amount of polysomes even under nutritional shift-down as examined using S. lividans strains (Fig. 4
).
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DISCUSSION |
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The gene cluster responsible for the production of Act has been cloned and characterized. Linked to the gene cluster, a pathway-specific regulatory gene (actII-ORF4) was shown to regulate the Act biosynthesis gene by acting as a positive regulator (Fernandez-Moreno et al., 1991). The expression of actII-ORF4 at the transcription level commences at the late growth phase in response to nutrient limitation (Chakraburtty & Bibb, 1997
). We have demonstrated that the K88E mutants of S. coelicolor and S. lividans both display a remarkable increase in the production of ActII-ORF4 protein at the late growth phase as determined by Western blot analysis (Hu & Ochi, 2001
; S. Okamoto & K. Ochi, unpublished results). When active protein synthesis is sustained under amino acid starvation (e.g. late growth phase), it would be highly advantageous for the production of proteins from newly transcribed genes (such as those involved in antibiotic production) at the late growth phase. Thus, the aberrant protein synthesis found in the K88E and P91S mutants could be the cause (solely or in part) of the remarkably activated antibiotic production of these mutant strains.
All the rpsL mutations found so far that confer resistance to streptomycin are located in two conserved regions: region I (41T to 47S) and region II (83R to 94R), each of which consists of characteristic loop structures in the S12 protein as demonstrated by the structural study of the Thermus thermophilus ribosome (Schluenzen et al., 2000; Wimberly et al., 2000
; Yusupov et al., 2001
). Region I has been reported to interact directly with the space between the 16S rRNA 530 loop and the 14921493 strand of the decoding site (Yusupov et al., 2001
). Most of the mutations in this region, found previously in E. coli and other bacteria, can increase translational accuracy. These mutations could have the effect of widening the space between the tRNAmRNA complex and the 30S A-site (Yusupov et al., 2001
). On the other hand, only a limited amount of data is available concerning the role of region II, although certain mutations found in this region caused an increase in translational accuracy (Table 2
). It is important to point out that the mutations K88E and P91S conferring an enhanced Act production are both located in region II but not region I.
The most prominent difference that discriminated between Act-overproducing rpsL mutants and other mutants came from amino acid shift-down experiments (Fig. 5). Despite the similar RNA synthesis profile upon shift-down, the K88E (and also P91S) mutant demonstrated an active protein synthesis, in agreement with the results from growing cultures (Fig. 2
). The effect of amino acid limitation on bacterial physiology has been well studied in both E. coli and Streptomyces spp. (Cashel et al., 1996
; Ochi, 1987
). Upon amino acid starvation, the level of uncharged tRNA increases, leading to activation of ppGpp synthetase (RelA). The increase in the ppGpp pool results in an immediate cessation of RNA accumulation and of other cellular reactions. The K88E mutation can reactivate Act production even in the genetic background of relA and relC (Ochi et al., 1997
; Shima et al., 1996
), which normally abolishes Act production by blocking ppGpp synthesis (Chakraburtty & Bibb, 1997
; Martinez-Costa et al., 1996
; Ochi, 1990
). Since active protein synthesis was detected upon amino acid shift-down of K88E mutants with genetic background of relA (Fig. 5
) and relC, ppGpp itself is not required for the activation of protein synthesis found in K88E mutants. Amino acid starvation in E. coli causes more than 90 % reduction in protein synthesis (O'Farrell, 1978
; Sorensen et al., 1994
), possibly by a limited availability of charged tRNA. In fact, the level of charged tRNA decreased 15- to 40-fold after starvation (Sorensen, 2001
). Jones (1977)
reported that there is a 70 % reduction of protein synthesis in ribosomes from late-growth-phase cells of Streptomyces antibioticus as determined by in vitro translation assay. Our observation that the K88E mutant ribosome remained in the form of polysomes at the late growth phase (Fig. 3
) or after amino acid shift-down (Fig. 4
) suggests that a large population of unstalled polysomes exists in the mutant cells.
An intriguing observation on structural changes of the ribosome was reported using cryo-electron microscopy, which clearly demonstrated the existence of a large space between two subunits in amino-acid-starved ribosomes (Ofverstedt et al., 1994; Zhang et al., 1998
). Since this structural change occurs immediately after amino acid depletion and is not seen during normal elongation equilibrium, it is possible that such an unusual open structure causes the reduction in translation rate. In the framework of this notion, our K88E mutant ribosome may also maintain the normal structure under amino acid starvation, because the increased stability was demonstrated under low-Mg2+ conditions (Table 3
). The region I loop of the S12 protein faces the top of the penultimate stem of 16S rRNA (at positions A1492 and A1493) and the switch helix (at positions 910 to 912) (Yusupov et al., 2001
). Positions A1492 and A1493 are directly involved in the interaction of tRNA at the A-site; tRNA binding and subunit association are somewhat coupled (Toivonen et al., 1999
). The helix (H44) comprising 1409 to 1491 and its counterpart helix (H69) in 23S rRNA, which links RNA molecules to one another, are suggested to play a role in subunit association (Yusupov et al., 2001
). Thus, it is likely that the K88E and P91S mutations in the S12 protein affected the interactions between 16S rRNA and 23S rRNA, eventually leading to an increased stability of the 70S complex.
It is well known that the translational error frequencies increase when cells are subjected to amino acid starvation (for a review, see Parker, 1989). The K88E mutant ribosome from the late-stationary-phase cells of S. coelicolor, however, displayed fourfold lower frequency of translational error than the wild-type ribosome (see Results). The accuracy of protein synthesis in the K88E mutant, though not a direct cause for the activation of antibiotic production, could enhance the cellular reactions such as antibiotic production that occur at the late growth phase.
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ACKNOWLEDGEMENTS |
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Received 15 May 2003;
revised 16 July 2003;
accepted 30 July 2003.
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