An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2)

Yoshiko Okamoto-Hosoya, Takeshi Hosaka and Kozo Ochi

National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

Correspondence
Kozo Ochi
kochi{at}affrc.go.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.


Abbreviations: Act, actinorhodin; Red, undecylprodigiosin


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomycetes are Gram-positive soil bacteria, which produce a wide variety of secondary metabolites such as antibiotics. Streptomyces coelicolor A3(2), and its close relative Streptomyces lividans 66, are the genetically best-studied strains in this genus; the complete genome sequence of S. coelicolor A3(2) is now available (Bentley et al., 2002). These strains have been used as models for studying the regulatory mechanisms of antibiotic production (reviewed by Champness & Chater, 1994; Chater & Bibb, 1996; Hopwood et al., 1994). S. coelicolor A3(2) produces several chemically distinct classes of antibiotics, two of which, namely actinorhodin (Act) and undecylprodigiosin (Red), are pigmented; S. lividans 66 normally does not produce antibiotics, even though it harbours a whole set of genes for the production of the antibiotics (Kieser et al., 2000).

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 {beta}-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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
The strains of S. coelicolor A3(2) and S. lividans 66 used are listed in Table 1. Fresh spore suspension was prepared from a culture grown at 30 °C on SFM agar medium (Kieser et al., 2000) and inoculated directly into YEME medium (Kieser et al., 2000). For amino acid shift-down experiments, the spore suspension was inoculated into 100 ml CD medium (Ochi et al., 1997) supplemented with 3 % vitamin-free Casamino acids (Difco). At the indicated times, cells were collected by filtration, washed with CD medium and resuspended in fresh CD medium with or without Casamino acids. Complementation testing of the P91S rpsL mutant with a plasmid harbouring the wild-type rpsL gene was performed as described by Kieser et al. (2000).


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Table 1. Strains of S. coelicolor A3(2) and S. lividans 66 used

 
Assay of actinorhodin and nucleotide pools.
Actinorhodin was measured as described by Kieser et al. (2000). Intracellular concentrations of GTP and ppGpp were measured as described by Ochi (1987), using HPLC on a Hitachi LaChrom Liquid Chromatography System with a Partisil-10 SAX (4·6x250 mm; GL Sciences) column.

[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 2–4 °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 (15–45 %) 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 (10–40 %) in the same gradient buffer.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
rpsL mutants of S. coelicolor and S. lividans are error restrictive
Although several str mutants were previously isolated in our laboratory or by Kieser et al. (see Table 1), we attempted in the present study to isolate other types of str mutants from the wild-type strains of S. coelicolor and S. lividans, which developed on GYM agar (Ochi, 1987) containing 10 or 50 µg streptomycin ml-1. Seventy mutants were randomly selected and subjected to rpsL gene sequencing. Eventually, we found three new mutant forms of rpsL (K43T, K43R and K88R) in S. coelicolor and four (K43N, K43T, K43R and K88R) in S. lividans. Representative strains of each mutant form are designated in Table 1 and were used for further study. These mutants as well as the previously isolated str mutants all showed a single amino acid substitution in ribosomal protein S12. As summarized in Table 1, four mutant forms (K43N, K43T, K43R and K88R) had no effect on antibiotic production, whereas two (K88E and P91S) enhanced (more than fivefold) or activated significantly the production of Act. Thus, the K88E mutant falls into the ‘P91S’ category and not into the second group as exemplified by K43N, K43T, K43R and K88R. The K88E mutation was previously demonstrated by gene replacement experiments to be responsible for the phenotype of high Act production (Shima et al., 1996). To demonstrate responsibility of the P91S mutation for the observed phenotype, we conducted a complementation test. When the strain KO-347 (P91S) was transformed with the low-copy-number plasmid pVWT (Okamoto-Hosoya et al., 2003), which harbours the wild-type rpsL gene, the resulting transformants had completely lost the ability to produce a high level of Act (data not shown), thus demonstrating responsibility of P91S mutation for Act overproduction. K88E and P91S (but not K43N and K88R) mutants exhibited a threefold increase in resistance (MIC 100 µg ml-1) to kirromycin. The rpsL mutants described above all grew well at rates comparable to those of the parental strains (Table 2).


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Table 2. Growth and in vitro translation characteristics of various str mutants

 
To investigate the presence or absence of the causal relationship between the translational accuracy level and the observed antibiotic overproduction, we determined the in vitro translation activity and translational accuracy using poly(U) and ribosome mixtures prepared from each mutant strain grown to mid-exponential phase (see Methods). As summarized in Table 2, all the rpsL mutants from S. coelicolor tested revealed a markedly increased translational accuracy (i.e. decrease in missense error rate). In particular, mutations K88E and K43N were most restrictive, while P91S was less restrictive. The increased translational accuracy in the K88E mutant was confirmed using ribosome mixtures prepared from cells grown to stationary phase [missense error rate of 0·0025 (K88E mutant) versus 0·0095 (wild-type strain 1147)]. It is apparent from these results that the accuracy level of each mutant strain is not correlated with the Act overproduction found in certain rpsL mutants.

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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Fig. 1. Growth of S. coelicolor A3(2) and S. lividans 66 compared with their rpsL (K88E) mutants. Cells were grown in YEME medium at 30 °C. (a) S. coelicolor wild-type strain 1147 ({circ}) and K88E mutant KO-178 ({bullet}); (b) S. lividans wild-type strain TK21 ({circ}) and K88E mutant TK24 ({bullet}).

 
We also compared the cell physiologies of the parental and mutant (K88E) strains. When 1147 (wild-type) and KO-178 (K88E) strains were grown in YEME medium, both accumulated ppGpp [2·5–5 pmol (mg dry wt)-1] temporarily at the point of growth cessation (36 h), accompanied by an abrupt decrease of GTP content [from 3000 pmol to 700 pmol (mg dry wt)-1]. Thus, the K88E mutant responds normally to nitrogen starvation despite its aberrant growth characteristics.

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 (45–51 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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Fig. 2. Incorporation of labelled leucine into the total protein fractions of cells at different growth phases. Cells were grown in YEME medium. At the indicated time, [3H]leucine was added to the culture and the incorporation of radioactivity into the acid-precipitable portion was measured for 30 min at 5 min intervals. The bars represent the rate of incorporation [c.p.m. min-1 (mg cell)-1]. (a) S. coelicolor wild-type strain 1147 (open bars) and K88E mutant KO-178 (filled bars); (b) S. lividans wild-type stain TK21 (open bars) and K88E mutant TK24 (filled bars). The data in the figure represent the means of two measurements.

 


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Fig. 3. Polysome profiles of S. coelicolor wild-type (1147) (a) and K88E mutant KO-178 (b) in different growth phases. Cells were grown in YEME medium for the indicated time. Polysome analysis is described in Methods.

 


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Fig. 4. Polysome profiles of S. lividans wild-type (TK21) (a) and K88E mutant TK24 (b) after amino acid shift-down. Cells were grown to mid-exponential phase in CD medium supplemented with 3 % Casamino acids and transferred into fresh CD medium without Casamino acids, followed by further 15 min incubation. Polysome analysis is described in Methods.

 
Comparison of translation activity after amino acid deprivation
Nutritional shift-down is one of the typical stress-provoking approaches used to analyse physiological changes within a short time (Ochi, 1987). We used amino acid shift-down to further discriminate the wild-type and mutant strains. S. coelicolor wild-type strain 1147 and its rpsL mutants 1258 (K43N), KO-482 (K88R), KO-178 (K88E) and KO-347 (P91S) were grown to mid-exponential phase in CD medium supplemented with 3 % Casamino acids, filtered, and transferred to CD medium lacking Casamino acids but containing [3H]leucine (to assay protein synthesis) and [14C]uracil (to assay RNA synthesis). The results of RNA and protein synthesis after Casamino acids deprivation are presented in Fig. 5(a) and (b), respectively. The RNA synthesis was severely suppressed immediately after amino acid depletion in all strains tested, representing the so-called ‘stringent response’ as reported previously for various Streptomyces spp. (Ochi, 1986; Strauch et al., 1991). In contrast, the behaviour of mutant strains was crucially discriminative in protein synthesis. Mutants 1258 (K43N) and KO-482 (K88R), together with the wild-type strain 1147, showed a low level of protein synthesis after amino acid depletion, but the capacity of mutants KO-178 (K88E) and KO-347 (P91S) for protein synthesis was fivefold greater (Fig. 5). The measurement of protein synthesis before shift-down was severely hampered because of the large amount of unlabelled leucine present in the medium. Thus, antibiotic-overproducing mutants (K88E and P91S mutants) were clearly discriminated from non-overproducing mutants (K43N and K88R) under amino-acid-limited conditions. The observed high activity of protein synthesis in K88E mutants was independent of ppGpp as the K88E effect could be detected even in the genetic background of relA (Fig. 6b) and relC (not shown), which caused a relaxed response with respect to RNA synthesis (Fig. 6a).



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Fig. 5. Incorporation of [14C]uracil and [3H]leucine into the total RNA (a) and protein fraction (b) after amino acid shift-down. Strains of S. coelicolor were grown in CD medium supplemented with 3 % Casamino acids and transferred into fresh CD medium with ({circ}) or without ({bullet}) Casamino acids. Incorporation of [3H]leucine and [14C]uracil was measured as acid-precipitable material.

 


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Fig. 6. Incorporation of [14C]uracil and [3H]leucine into the total RNA (a) and protein (b) fraction after amino acid shift-down in S. coelicolor relA mutants. Experimental procedures were the same as for Fig. 5. {circ}, With Casamino acids; {bullet}, without Casamino acids.

 
The K88E mutant ribosome forms a more stable 70S complex
The topography of amino acid-‘starved’ ribosomes has been studied previously by others in E. coli using the electron microscope. Interestingly, deprivation of an essential amino acid induced a spatial separation of the subunits in the ribosome, indicating that such ribosome is more open than that from growing cells (Ofverstedt et al., 1994; Zhang et al., 1998). This open structure appears to be specific to the ribosomes that were starved for amino acid, since no gross morphological changes of 70S structure has been reported between the pre- and post-translocational states (Noller et al., 1990; Stark et al., 1997). It was also reported that ribosomes from methionine-starved cells are structurally unstable in the presence of low concentrations of Mg2+ (Sells & Ennis, 1970), suggesting a structural instability of open-form ribosomes. However, there are no reports on the use of a mutant ribosome for studying the ribosomal structure or structural stability. In the present study, we reasoned that the ribosomes of our mutants (K88E and P91S) could maintain more stable and closer inter-subunit structure, thus leading to active protein synthesis even under amino acid-starved conditions. To assess this hypothesis, we prepared ribosomes from both wild-type and mutant strains of S. coelicolor and S. lividans, and examined the stability of the 70S complex in the presence of various concentrations of Mg2+. The dissociation pattern of 70S ribosomes to 30S and 50S subunits at varying concentrations of Mg2+ and the results of the density-gradient analysis are presented in Table 3 and Fig. 7 respectively. The 70S ribosome was stable at concentrations of Mg2+ higher than 3 mM; however, it dissociated to 30S and 50S subunits when the concentration of Mg2+ was decreased to less than 2 mM. Strikingly, the ribosomes of K88E and P91S mutants, from strains KO-178 and KO-347 respectively, displayed a greater fraction of 70S particles than the wild-type strain at all Mg2+ concentrations tested, but particularly at low concentrations (1–2 mM). In contrast, mutant ribosomes from strain 1258 (K43N) showed a somewhat reduced stability. The increased stability of the K88E mutant ribosome was also demonstrated in S. lividans (Table 3). Thus, the Act-overproducing mutants with an enhanced protein synthesis were characterized by a stable 70S complex.


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Table 3. Ratio of 70S ribosome particles in the presence of various concentrations of Mg2+

 


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Fig. 7. Sucrose gradient analysis of ribosome fractions prepared from growing cultures of S. coelicolor wild-type and K88E mutant KO-178. Procedures are described in Methods. The analysis was performed at a MgCl2 concentration of 1 mM.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have previously reported that antibiotic production by bacteria, including Streptomyces spp., is dramatically activated by introducing certain mutations into the rpsL gene that confer resistance to streptomycin (Hosoya et al., 1998; Hu & Ochi, 2001; Shima et al., 1996). Our principal findings in this study were that (i) the rpsL mutants with an enhanced Act production demonstrated a capacity for active protein synthesis even under amino acid starvation conditions, and (ii) the ribosomes from these rpsL mutants were structurally stable in low concentrations of Mg2+. These observations were supported by the fact that none of the rpsL mutants incapable of enhancing Act production had such aberrant characteristics.

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 1492–1493 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 tRNA–mRNA 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.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Organized Research Combination System (ORCS) of the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank A. Lezhava, S. H. Oh, T. Inagawa, J. Xu and S. Okamoto for performing several of the preliminary experiments. We also acknowledge Y. B. Ngwai for his comments on the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 15 May 2003; revised 16 July 2003; accepted 30 July 2003.



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