SummaryStreptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycinresistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers lowlevel streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.

A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization. Actinomycetes produce a variety of natural products that are of major importance in the pharmaceutical industry. More than 50% of all anti-infective and anticancer compounds developed over the past 25 years have been natural products or derivatives thereof (1). Discovery of novel antibiotics and strain improvement for overproduction are important in applied microbiology research, especially in the production of clinically important antibiotics as well as antibiotics important in veterinary medicine and agriculture. There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. That is, genome sequencing projects have revealed many biosynthetic gene clusters for the production of unknown secondary metabolites. For example, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, and Saccharopolyspora erythraea are each known to produce three to five secondary metabolites but actually possess Ͼ20 clusters that encode known or predicted biosynthetic pathways for secondary metabolites (2-5). Exploitation of such genetic potential in actinomycetes may lead to the isolation of new biologically active compounds (6-8). We recently described a new method to increase antibiotic production in bacteria by modulating ribosomal components (ribosomal proteins or rRNA), i.e., by introducing mutations conferring drug resistance, as many antibiotics target the ribosome (9-11). This new approach, called "ribosome engineering" (12, 13), has several advantages, including the ability to screen for drug resistance mutations by simple selection on drugcontaining plates, even if the mutation frequency is extremely low (e.g., Ͻ10 Ϫ10 ), and the ability to select for mutations without prior genetic information. Hence, this method requires no induced mutagenesis. Interestingly, the introduction of several drug resistance mutations has a cumulative effect on antibiotic production (14-16).In addition to enhancement of antibiotic production, we have demon...

Certain streptomycin resistance mutations (i.e., rpsL and rsmG) result in the overproduction of antibiotics in various actinomycetes. Moreover, rpsL rsmG double-mutant strains show a further increase in antibiotic production. rpsL but not rsmG mutations result in a marked enhancement of oligomycin production in Streptomyces avermitilis and erythromycin production in Saccharopolyspora erythraea, accompanied by increased transcription of a key developmental regulator gene, bldD, in the latter organism.Recently, we described a practical method of increasing antibiotic production in bacteria by modulating ribosomal components (ribosomal proteins or rRNA), specifically by the introduction of mutations conferring drug resistance (5,22,23). This approach, called "ribosome engineering" (12, 14), has several advantages, including the ability to screen for drug resistance mutations by simple selection on drug-containing plates, even if the mutation frequency is extremely low (e.g.,Ϫ10 ). As demonstrated with Streptomyces coelicolor A3(2) and Streptomyces albus, ⌬rsmG and rpsL(K88E) mutants exhibit enhanced protein synthetic activity during the late growth phase (10,16,21), which is consistent with our hypothesis (4)

Genome sequencing projects have revealed many biosynthesis gene clusters for the production of as-yet unknown secondary metabolites, especially in actinomycetes. Here, we report that the rare earth elements, scandium and/or lanthanum, markedly activate, ranging from 2.5-to 12-fold, the expression of nine genes belonging to nine secondary metabolite-biosynthetic gene clusters of Streptomyces coelicolor A3(2) when added to the medium at low concentrations. HPLC analysis of ethyl acetateextractable metabolites indicated the detectability of several compounds only in the rare earth-treated cultures. This approach should facilitate discovery of new biologically active compounds and the study of secondary metabolite production.

Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

e Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the bluepigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F o F 1 ATP synthase ␣ and ␤ subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed. Streptomyces is the largest genus of Gram-positive filamentous actinomycetes, and members of this genus produce abundant amounts of numerous bioactive metabolites, including antitumor agents, immunosuppressants, and antibiotics in particular (1, 2). Whole-genome sequencing studies of Streptomyces strains have shown that each species could produce many more secondary metabolites than were expected. For example, Streptomyces coelicolor A3(2), S. avermitilis MA-4680, and S. griseus IFO 13350 each produce several secondary metabolites, although they have more than 20 gene clusters that can encode a number of known or predicted biosynthetic pathways for secondary metabolites (3-5). This indicates that the vast majority of secondary metabolites remain unexpressed or barely expressed under standard laboratory conditions. Thus, there is considerable interest in exploring practical means to induce this genetic potential in streptomycetes, which could result in the isolation of novel bioactive secondary metabolites.Recent developments in new methodologies, including physiological and genetic engineering approaches, have opened the door for the discovery of novel secondary metabolites by activating cryptic biosynthetic pathways in streptomycetes (6-13). The improvements and modifications of ...

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The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10 ؊6 . More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA ؉ strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tsp r and Rif r ), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic-failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.