Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

We recently described a new method to activate antibiotic productionin bacteria by introducing a mutation conferring resistanceto a drug such as streptomycin, rifampin, paromomycin, or gentamicin.This method, however, enhanced antibiotic production by onlyup to an order of magnitude. Working with Streptomyces coelicolorA3(2), we established a method for the dramatic activation ofantibiotic production by the sequential introduction of multipledrug resistance mutations. Septuple and octuple mutants, C7and C8, thus obtained by screening for resistance to seven oreight drugs, produced huge amounts (1.63 g/liter) of the polyketideantibiotic actinorhodin, 180-fold higher than the level producedby the wild type. This dramatic overproduction was due to theacquisition of mutant ribosomes, with aberrant protein and ppGppsynthesis activity, as demonstrated by in vitro protein synthesisassays and by the abolition of antibiotic overproduction withrelA disruption. This new approach, called "ribosome engineering,"requires less time, cost, and labor than other methods and maybe widely utilized for bacterial strain improvement.

INTRODUCTION

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INTRODUCTION

Strain improvement is important in applied microbiological research,especially in the production of clinically important antibioticsas well as antibiotics important in veterinary medicine andagriculture. Current methods of antibiotic production, rangingfrom classical random approaches to metabolic engineering, areeither costly or labor-intensive. We recently described a newmethod to increase antibiotic production in bacteria by modulatingribosomal components (ribosomal proteins or rRNA), i.e., bythe introduction of mutations conferring drug resistance, sincemany antibiotics target the ribosome (13, 29, 31). This newapproach, called "ribosome engineering" (28), has several advantages.These advantages include the ability to screen for drug resistancemutations by simple selection on drug-containing plates, evenif the mutation frequency is extremely low (e.g., <10^–10),and the ability to select for mutations without prior geneticinformation. Thus, this method requires no induced mutagenesis.This approach has been used to enhance the production of salinomycinin an industrial strain of Streptomyces albus (33); to activatethe synthesis of dormant antibiotics (15, 19); to improve chemicaltolerance in Pseudomonas putida (12), a valuable bacterium forwaste processing; and to enhance the synthesis of enzymes suchas ${alpha}$ -amylase in Bacillus subtilis (22). Interestingly, the introductionof several drug resistance mutations had a cumulative effecton antibiotic production. This was shown by the sequential introductionof three drug resistance mutations into Streptomyces coelicolorA3(2) (str, gen, and rif, which confer resistance to streptomycin[Sm], gentamicin [Gen], and rifampin [Rif], respectively) andthree rounds of selection, with the resulting triple mutant,SGR, showing hierarchical increments of antibiotic production(14).

S. coelicolor A3(2), the genetically best-characterized strainof Streptomyces, produces at least four distinct classes ofantibiotics (21), including the blue-pigmented polyketide antibioticactinorhodin (Act), thus providing an easily tractable systemfor the methodological study of strain improvement. Based onour previous results using single mutations or multiple mutations,we demonstrate here the efficacy of septuple and octuple drugresistance mutations for the dramatic activation of antibioticproduction. The mechanisms underlying this remarkable activationwere also studied.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Drugs and chemicals.
Geneticin, paromomycin (Par), fusidic acid (FA), lincomycin(Lin), and thiostrepton (Tsp) were purchased from Sigma; Smwas purchased from Nacalai Tesque, Inc. (Japan); and Gen, Rif,tetracycline (Tet), and hygromycin were purchased from WakoPure Chemicals (Japan). The standards guanosine tetraphosphate(ppGpp) and guanosine pentaphosphate (pppGpp) were preparedenzymatically in our laboratory, each with a purity of >95%.

Bacterial strains and preparation of multiply drug-resistant mutants.
S. coelicolor A3(2) wild-type strain 1147, triple mutant strainSGR (14), and its multiply drug-resistant derivatives are listedin Table 1. All of the mutations arose spontaneously, and noinduced mutagenesis was required. For each experiment, strainswere inoculated onto GYM (31) or SFM (21) plates and incubatedfor approximately 7 to 10 days for sporulation. Fresh sporesuspensions were inoculated into 100 ml medium in a 500-ml flaskand incubated on a rotary shaker (200 rpm) at 30°C exceptwhere indicated.

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TABLE 1. S. coelicolor A3(2) and its drug-resistant derivatives used in this study

Triple mutant strain SGR (14) was used as the starter strainto select for multiple-drug-resistant mutants, with generalscreening procedures performed as described previously (28).The procedure used to prepare multiply drug-resistant mutantsis illustrated in Fig. 1A. Spore suspensions (usually >10⁹spores) were spread and incubated for 7 days. Antibiotic-resistantclones were first screened for the production of Act on platecultures, with candidates having the deepest blue color beingassessed for Act production assays using liquid media. If necessary,mutations were mapped by DNA sequencing. In each step, one ortwo colonies with the highest Act production were used for thenext round of screening. SGR spore suspensions were spread ontoGYM plates containing various concentrations of Par, usuallyapproximately 3- to 30-fold of the parental strain's MIC toselect for Par resistance, followed by similar steps of screeningfor resistance to Geneticin, FA, Tsp, and Lin, finally yieldingoctuple mutants with resistance to eight different drugs.

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FIG. 1. Act production and actII-ORF4 transcription in drug-resistant mutants. (A) Illustration of the procedure used for the sequential introduction of drug resistance mutations to generate multiple-drug-resistant mutants. (B) Act production on agar plates. Strains were inoculated onto GYM plates, which were incubated for 8 days. The reverse sides of the plates are shown to illustrate the blue antibiotic Act. (C) Act production in liquid culture. Strains were inoculated into liquid GYM or GYM33 medium and incubated on a rotary shaker (200 rpm) for 8 days. Act production by each strain was measured as the maximum OD₆₄₀ value. (D) Transcriptional analysis of actII-ORF4 by RT-PCR. Total RNA preparation and RT-PCR were performed as described in Materials and Methods. Eight microliters of each PCR product was loaded for electrophoresis. The gel profile of rRNA (4 µg per lane) is presented as a reference. (E) Transcriptional analysis of actII-ORF4 by real-time qPCR. Total RNA preparation and real-time qPCR were performed as described in Materials and Methods. Each transcriptional assay was normalized to that of hrdB. The error bars indicate the standard deviations of the means of triplicate samples.

Antibiotic production assay.
GYM, R2YE (21), R5 (21), and R3 (31) media were used to screenfor the production of the blue antibiotic Act on agar platesby directly assessing the density of the blue color. For Actproduction in liquid media, culture samples (0.5 ml) were mixedwith equal volumes of 2 M KOH, vortexed, and centrifuged at3,000 x g for 5 min. The Act concentration in the supernatantswas determined by measuring the absorbance at 640 nm (opticaldensity at 640 nm [OD₆₄₀]; ${varepsilon}$ = 25,320 for the pure compound)(21). Red in liquid GYM medium and calcium-dependent antibiotic(CDA) on plates were assayed as described previously (21). Forantibiotic production, three flasks were always used for eachstrain, with production confirmed by at least two separate experiments.

Determination of MICs.
To determine MICs, spore solutions were dotted onto GYM platescontaining various concentrations of a drug and incubated at30°C for 48 h, with the minimum drug concentration ableto fully inhibit growth defined as the MIC.

Mutation analysis.
Primers used to amplify the candidate DNA fragments are listedin Table S1 in the supplemental material. PCR amplificationwas carried out with ExTaq (Takara). Purified PCR products weredirectly sequenced with BigDye Terminator cycle sequencing kits(Perkin-Elmer Applied Biosystems, Foster City, CA). The sequencedata were aligned using the GENETIX program (Software DevelopmentCo., Tokyo, Japan).

Disruption of relA.
S. coelicolor A3(2) is characterized by high-frequency conjugationdue to the presence of the sex factor plasmid SCP1 (21). Toknock out the relA gene in the octuple mutant C8, conjugationwas performed using the relA null mutant M570 (5), in whichmost of the relA gene (corresponding to amino acid residues167 to 683 of 847 residues) was replaced by a hygromycin resistancegene (hyg). Strains C8 and M570 were mix cultured on a R2YEplate, and the spores that formed after 5 days were spread ontoa selection plate containing 5 µg/ml of hygromycin andall eight drugs. The conjugant (C8relA2) thus obtained was assayedby PCR for the absence of the internal segment of relA and thepresence of hyg.

Analysis of gene transcription.
Total RNAs were purified from cells grown on GYM plates coveredwith cellophane for the indicated times using Isogen reagent(Nippon Gene) according to the manufacturer's protocol. Aftertreatment with RNase-free DNase I (amplification grade; Invitrogen),1 µg of each of the total RNAs was used as a templatefor reverse transcription (RT) (20 µl) with a ThermScriptRT-PCR kit (Invitrogen). Primers used for RT-PCR are listedin Table S1 in the supplemental material. The amount of RT productsused as a PCR template and numbers of PCR cycles were optimizedfor each gene. In a 50-µl PCR mixture, 1 and 2 µlof reverse transcript products were used for actII-ORF4 andhrdB (encoding the major sigma factor), respectively, whichwere amplified with 26 cycles, and 4 µl was used for relAfor 28 cycles.

Real-time quantitative PCR (qPCR) analysis of gene transcriptionwas conducted using the 7300 real-time PCR system and Sybr greenPCR master mix (Applied Biosystems) as described previouslyby Kasai et al. (20), except that random hexamers included inthe ThermScript RT-PCR kit (Invitrogen) were used in the RTreaction and except that the annealing temperature of qPCR was60°C. The transcription of hrdB, a gene encoding the principalsigma factor of RNA polymerase, was used as the internal control.Each transcriptional assay was normalized to the correspondingtranscriptional level of hrdB. Primers used for real-time qPCRare listed in Table S1 in the supplemental material.

Preparation of ribosomes and the S-150 fraction.
Ribosomes and the S-150 fraction (i.e., the supernatant followingcentrifugation at 150,000 x g for 3 h) were prepared from cellsharvested at various growth phases in YEME medium (21), as describedpreviously (11), except that cells were first washed twice witha high-salt buffer containing 1 M (instead of 30 mM) ammoniumacetate before washing with the standard buffer and except that2 mM (instead of 1 mM) phenylmethylsulfonyl fluoride was includedin all buffers.

In vitro protein synthesis.
Cell-free synthesis of green fluorescent protein (GFP) was performedas described previously (11). In brief, 20 A₂₆₀ units/ml ofribosome or 0.5 mg/ml of the S-150 fraction was incubated at30°C for 15 min in a 100-µl total volume of a mixturecontaining 1.2 mM ATP, 0.8 mM GTP, 0.64 mM cyclic AMP, 15 µgEscherichia coli total tRNAs, and 0.4 mM each of the 20 naturalL-amino acids. GFP synthesis was initiated by the addition of100 µg gfp mRNA, which had been synthesized in vitro froma plasmid gfp gene using T7 RNA polymerase (Takara). Aliquots(10 µl) were withdrawn every 30 min, electrophoresed onnative 10% polyacrylamide gels, and subjected to fluorescenceanalysis using a FluoroImager (Molecular Dynamics).

Assay of ppGpp.
Intracellular ppGpp was extracted from cells cultured on GYMand on modified R5 (16) plates covered with cellophane with1 M formic acid and assayed by high-performance liquid chromatography(27). For nutritional shift down, exponentially growing cellsin chemically defined (CD) medium (27) supplemented with 3%Casamino Acids (vitamin free; Difco) were rapidly transferredinto fresh CD medium without Casamino Acids. Cells were harvested0, 15, 30, and 60 min after cell transfer for the extractionof ppGpp; pppGpp was not detectable in these samples.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Construction of multiply drug-resistant mutants.
Starting with triple mutant strain SGR (14), we screened forPar resistance by spreading SGR spores onto GYM plates containingvarious concentrations of Par, as illustrated in Fig. 1A. About20% of the colonies that developed within 7 days due to spontaneousmutations showed increased Act production compared with thatof parental strain SGR. Since we previously found that a Parresistance mutation (P91S) in ribosomal protein S12 effectivelyresulted in Act overproduction (29), we sequenced the S12-encodinggene, rpsL, in SGR and dozens of Act-overproducing colonies.As expected, we found that in addition to the rpsL-encoded mutationK88E present in the SGR mutant, all colonies tested possesseda second amino acid substitution in S12, including the P91Smutation. To our surprise, we found that one colony containeda novel Par resistance mutation consisting of an inserted glycineresidue (G, encoded by ggg) at position 92 of S12 (92::G), althoughit appeared at a relatively low frequency. More interestingly,this new insertion mutation plus K88E rendered cells able toproduce even more Act than P91S combined with K88E. The introductionof 92::G (and P91S) (not shown) increased the sensitivitiesof mutants to many antibiotics, including Geneticin, FA, Lin,and Tet (Table 2). Consequently, a quadruple mutant, SGRP, containing92::G was used for further selection.

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TABLE 2. Levels of resistance of S. coelicolor A3(2) strains to various drugs

Subsequent sequential screenings were performed to isolate strainsthat were resistant to Geneticin, FA, Tsp, and Lin, generatingquintuple, sextuple, septuple, and octuple mutants (C5, C6,C7, and C8, respectively) (Table 1). During these screenings,we found that as many as 5% to 15% of the mutant strains producedmore Act than the corresponding parental strain. The periodrequired for all screenings was 8 months. In general, as thenumber of mutations increased, the growth rate slowed (see Fig.3A), a finding consistent with the opinion that drug resistancewas often obtained at the cost of growth fitness (1).

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FIG. 3. Growth patterns and in vitro protein synthesis activities of wild-type strain 1147, triple mutant strain SGR, and octuple mutant strain C8. (A) Growth profiles. Spores were inoculated into liquid YEME medium, with absorbance at 450 nm (OD₄₅₀) monitored for the growth curve. An OD₄₅₀ of 0.06 was defined as the zero time point approximately 16 to 24 h after inoculation. Arrows indicate the time points at which cells were harvested for preparation of ribosomes and the S-150 fraction. (B) In vitro GFP synthesis was assayed using ribosomes from 1147 ( ${blacksquare}$ ), SGR ( ${square}$ ), and C8 ( ${square}$ ), with nascent gfp mRNA as the template. Top, fluorographs. Bottom, fluorescence intensities of bands after 90 min of reaction, as determined by scanning the fluorographs.

Octuple mutant strain C8 was somewhat temperature sensitive;wild-type strain 1147 grew at 40°C, but C8 did not growat 39°C. In addition, C8 exhibited extremely slow growthin a chemically defined medium. In liquid culture, mutant strainC8 showed a tight "core" pellet with short-fragmented mycelia,whereas the wild type (1147) possessed a core surrounded bymany naturally stretching, branched mycelia (Fig. 2). Importantly,mutant strain C8 was genetically stable; no obvious changesin morphology and Act productivity were found even after 16sequential inoculations onto GYM agar plates.

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FIG. 2. Morphological appearance of wild-type strain 1147 and octuple mutant strain C8 during exponential growth phase in liquid R2YE medium. Bars represent 20 µm.

Antibiotic overproduction and mutation analysis.
The relationship between Act production and the number of mutationsis shown in Fig. 1B and C. We found that C7 produced 0.51 g/literand that C8 produced 0.55 g/liter Act, 88- and 95-fold higher,respectively, than the 5.8 mg/liter produced by wild-type 1147in liquid GYM medium. It was noteworthy that low temperaturescould further enhance Act production by these multiply drug-resistantmutants. When strains were incubated at 27°C, C7 produced0.63 g/liter Act, and C8 produced 0.90 g/liter Act, or 24% and64% higher, respectively, than at 30°C. Increased Act productionwas more pronounced when mutants were cultivated in a densemedium, GYM33 (GYM medium supplemented with 3% starch and 3%soybean powder). In this medium, C7 and C8 produced 1.63 g/literand 1.22 g/liter Act, respectively, or 180- and 136-fold morethan the 9 mg/liter produced by wild-type strain 1147 (Fig.1C).

Transcriptional analysis by RT-PCR showed that the expressionof the actII-ORF4 gene, encoding the positive regulator of Actbiosynthesis (7), was greatly enhanced in drug-resistant mutants,especially in the late phase of mutants (C6 and C8) (Fig. 1D).This was confirmed by real-time qPCR, in which the expressionlevel of actII-ORF4 in the C8 mutant was sevenfold higher thanthat in wild-type strain 1147 (Fig. 1E). These results indicatethat drug resistance mutations exerted their effects on antibioticproduction at the transcriptional level. In addition to Act,these multiply drug-resistant mutants showed increased productivityof undecylprodigiosin (red) and CDA (data not shown), althoughthe observed increase (20- to 30-fold) was not as dramatic asthat of Act.

Structural studies on the mechanism of action of antibioticshave shown that many of these agents target ribosomal components(35, 36). For example, most FA resistance mutations are clusteredin the fusA gene, which encodes elongation factor G (3); Tspresistance is often due to mutations in or a deletion of theribosomal protein L11 (27, 30), and Lin resistance is frequentlydue to mutations in the ribosomal proteins S7, L14, and L15(17). Octuple mutant strain C8, however, did not have mutationsin the fusA gene, the L11-encoding gene rplK, and the genesencoding S7, L14, and L15. Apparently, new types of drug resistancemutations tend to appear in these "unnatural ribosomes" thatcontain multiple drug resistance mutations.

Mutant ribosomes sustain a high level of protein synthesis even at the late growth phase.
Although some mutations were not identified, it is likely thatribosomal properties and concomitant protein translation weremodified largely by drug resistance mutations, because all drugsused in this study, except rifampin, target ribosomal components.We therefore assessed the in vitro protein synthesis activitiesof the mutants using nascent GFP mRNA as a template. Ribosomaland S-150 fractions were prepared from wild-type 1147, triplemutant strain SGR, and octuple mutant strain C8 cells grownin YEME medium to mid-exponential phase (E), early stationaryphase (S2), and late stationary phase (S4) (Fig. 3A). GFP wasabundantly synthesized by ribosomes prepared from cells of allstrains in the E phase. Ribosomes prepared from wild-type cellsin S2 synthesized much less GFP, and ribosomes prepared fromwild-type cells in S4 lost their ability to synthesize protein.By contrast, ribosomes prepared from drug-resistant mutant strainsSGR and C8 in both S2 and S4 showed high levels of protein synthesisactivity (Fig. 3B). Of note, ribosomes prepared from the octuplemutant C8 during exponential and stationary phases maintainedrelatively constant levels of protein synthesis activity, decreasingonly about 25% upon entering stationary phase (Fig. 3B). Furthermore,ribosomes prepared from cells at an extremely late growth phase(S5) (Fig. 3A) had over 60% protein synthesis activity comparedwith ribosomes from E-phase cells (data not shown). Thus, theability of late-growth-phase cells to sustain a high level ofprotein synthesis activity appears to be a key feature of drug-resistantmutants, as we demonstrated previously (11), an activity especiallyimportant for the expression of stationary-phase-specific genes,including those for secondary metabolism.

Mutant cells acquire an increased ability to accumulate ppGpp.
Bacterial cells exert "stringent control" over a wide varietyof genes and enzymes when they encounter adverse environmentalconditions, such as the limited availability of an essentialnutrient. ppGpp is a key mediator of this response and is thereforecalled a bacterial alarmone (4). Experiments using knockoutmutants of genes necessary for ppGpp synthesis or forcing ppGppsynthesis under nutrient-sufficient conditions have indicatedthat ppGpp plays a pivotal role in the onset of antibiotic productionin bacteria, including Streptomyces spp. (5, 6a, 9, 26, 27)and B. subtilis (18).

Strikingly, when grown on GYM agar, the octuple mutant C8 hada much higher intracellular level of ppGpp (4.5 to 13 pmol/mg[dry weight]) than wild-type strain 1147 (0.3 to 1.6 pmol/mg[dry weight]) (Fig. 4A). The high level of ppGpp in C8 was especiallypronounced during the early growth phase (20 to 30 h), whenits ppGpp level was 10- to 30-fold higher than that in the wildtype. Similar results were obtained in liquid culture (see Fig.S1 in the supplemental material) and on modified R5 plates (datanot shown). The ability to accumulate a high level of ppGppwas conferred mainly upon the introduction of Tsp resistance,as measured by intracellular ppGpp levels of each drug-resistantmutant at 29 h (Fig. 4B). To clearly demonstrate the intrinsicrole of mutant ribosomes, the ability of the mutant to synthesizeppGpp was analyzed in shift-down experiments. In both wild-typeand mutant strains, ppGpp synthesis was activated immediatelyafter the depletion of amino acids, peaking 15 min after nutritionalshift down. As expected, we found that C8 synthesized threefoldmore ppGpp than did wild-type strain 1147 (Fig. 5), indicatingthat C8 mutant ribosomes synthesized and accumulated higherlevels of ppGpp.

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FIG. 4. Accumulation of intracellular ppGpp and transcriptional analysis of the relA gene in drug-resistant mutants. (A) Intracellular ppGpp levels of wild-type strain 1147, octuple mutant strain C8, and relA deletion mutant strain C8relA2 during growth on GYM plates. Cells were harvested at the indicated time points for ppGpp extraction. ${blacksquare}$ , 1147; ${square}$ , octuple mutant strain C8; ${triangleup}$ , relA deletion mutant strain C8relA2. (B) Intracellular ppGpp levels of each drug-resistant mutant at 29 h (corresponding to the arrow in A). The increase in the ppGpp level upon the introduction of Tsp resistance is indicated by an arrow. (C) Transcriptional analysis of relA in drug-resistant mutants by RT-PCR using the total RNA samples as described in the legend of Fig. 1D. Transcription of hrdB was utilized as an internal control. (D) Transcriptional analysis of relA by real-time qPCR. Total RNA preparation and real-time qPCR were performed as described in Materials and Methods. Each transcriptional assay was normalized to that of hrdB. The error bars indicate the standard deviations of the means of triplicate or more samples.

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FIG. 5. Intracellular ppGpp accumulation after nutritional shift down. After growth to mid-exponential phase in CD medium plus 3% Casamino Acids, cells were rapidly transferred into fresh CD medium without Casamino Acids, and ppGpp was extracted with 1 M formic acid at the indicated time points. The error bars indicate the standard deviations of the means of triplicate samples. ${blacksquare}$ , wild-type strain 1147; ${square}$ , octuple mutant strain C8.

Although the detailed molecular mechanisms underlying thesephenomena are at present not known, it is important that theexpression of the relA gene, which encodes ppGpp synthetase,was elevated remarkably at the transcriptional level in themutants, as shown by RT-PCR analysis (Fig. 4C) and as confirmedby real-time qPCR analysis, which displayed a 4.4-fold-elevatedlevel of transcription of the relA gene in mutant strain C8(Fig. 4D). It was also confirmed that these multiply drug-resistantmutants have no mutations in the relA gene. Several kinds ofdrug resistance mutations (including rif and fus mutations)were previously reported to disturb ppGpp synthesis (23, 24)but show a reduction in the ppGpp level. Our finding is thereforethe first to demonstrate the existence of mutations (especiallya tsp mutation) that cause an increase in ppGpp levels.

relA disruption abolishes a mutant's antibiotic overproduction.
To confirm the important role of ppGpp in the dramatic activationof Act production in multiply drug-resistant mutants, the relAgene was knocked out in octuple mutant strain C8 by conjugationwith relA null mutant strain M570 (5), which is entirely deficientin ppGpp synthesis. This conjugant, C8relA2, showed a completeinability to accumulate ppGpp throughout all growth phases (Fig.4A) as well as a >60% decrease in Act production (Fig. 1B and C),indicating the critical role of elevated ppGpp levels in theantibiotic overproduction observed in mutant strain C8. Theimpaired growth of mutant strain C8 was restored only partiallyby introducing the relA mutation (data not shown).

Recent analyses of the ppGpp system in plant cells (6, 32, 34)and X-ray structural analysis of the RNA polymerase-ppGpp complex(2) have enhanced our understanding of the physiological significanceof the stringent control mediated by ppGpp. Analysis of theeffect of ppGpp accumulation on changes in the S. coelicolorA3(2) transcriptome suggested a global role for ppGpp in cellularfunction (10). The induction of ppGpp synthesis activated thetranscription of genes for Act and CDA biosynthesis, a findingin good agreement with the results shown here.

Concluding remarks.
We have developed a more rapid and cost-effective and less labor-intensivemethod (i.e., ribosome engineering) for the dramatic activationof antibiotic production by constructing multiply drug-resistantmutants. Our results demonstrate that this dramatic overproductionof antibiotics was due to mutant ribosomes with aberrant proteinand ppGpp synthesis activities. These findings indicate thatribosomes can be good targets for strain improvement in bacteria,although it is still unclear why ribosomal mutations markedlyenhance relA transcription (Fig. 4C and D). We reported similarresults for metK (coding for S-adenosylmethionine synthetase),the expression of which was activated more than 30-fold by anrsmG mutation in S. coelicolor, which results in the failureto methylate 16S rRNA at position G518 (25). Clarification ofthese peculiar phenomena at the molecular level may open newhorizons for the study of still-unknown ribosomal functions.Although in the present study, we used mainly three strains(1147, SGR, and C8) as representatives to be characterized insome detail, more convincing evidence should come from a thoroughanalysis of each successive step in mutagenesis, since ribosomalalterations and altered ppGpp synthesis abilities do not necessarilyoccur together or occur in each successive mutant strain.

The present findings are in agreement with our proposal (11,28) that the cell's capacity to synthesize protein at a lategrowth phase is crucial for accelerating the initiation of onsetof secondary metabolism and for the production of abundant biosyntheticenzymes. The principal regulator of Act production in S. coelicolorappears to be the availability of the pathway-specific transcriptionalregulatory protein ActII-ORF4, a threshold concentration ofwhich is required for the efficient transcription of its cognatebiosynthetic structural genes (7). Although we do not yet knowhow the drug resistance mutations mediated preferential genetranscription work (Fig. 1D and E), it is conceivable that theexpression of pathway-specific regulatory genes (e.g., actII-ORF4and redD) is governed by higher-order regulatory proteins andthat the expression of the latter presumptive regulatory proteinsmay be significantly affected under conditions associated withenhanced protein synthesis during the stationary phase in themutants.

ACKNOWLEDGMENTS

This work was supported by grants to K.O. from the EffectivePromotion of Joint Research of Special Coordination Funds (Ministryof Education, Culture, Sports, Science, and Technology of theJapanese Government).

The relA null mutant M570 was a generous gift from Mervyn Bibbof the John Innes Institute.

FOOTNOTES

* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-29-838-8125. Fax: 81-29-838-7996. E-mail: kochi{at}affrc.go.jp

Published ahead of print on 29 February 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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