Yukinori Tanaka

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria.

Streptomycin 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 streptomycin‐resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low‐level 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 (m7G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so‐called 530 loop. Thus, we have identified gidB as a new streptomycin‐resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m7G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10−6 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.

Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements

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.

Activation and Products of the Cryptic Secondary Metabolite Biosynthetic Gene Clusters by Rifampin Resistance (rpoB) Mutations in Actinomycetes

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.

Rare earth elements activate the secondary metabolite–biosynthetic gene clusters in Streptomyces coelicolor A3(2)

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 acetate-extractable 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.

Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance

ABSTRACT 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 (Tspr and Rifr), 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.

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

ABSTRACT 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 blue-pigmented 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 (FoF1 ATP synthase α and β subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.

Activation of secondary metabolite-biosynthetic gene clusters by generating rsmG mutations in Streptomyces griseus

Unlike other Streptomyces spp., the streptomycin producer Streptomyces griseus IFO13189 shows emergence of a small fraction of rsmG and rpsL mutants among spontaneous low- or high-level streptomycin-resistant mutants. rsmG, but not rpsL, mutants showed greater ability (two- to threefold) to produce streptomycin, accompanied by enhanced transcription of metK and strR, together with streptomycin biosynthetic genes, such as strB1, strD and strF, thus underlying the observed increase in streptomycin production in the rsmG mutants. Moreover, rsmG mutation was effective for activating the ‘silent’ or poorly expressed secondary metabolite–biosynthetic genes present in S. griseus.

The mthA Mutation Conferring Low-Level Resistance to Streptomycin Enhances Antibiotic Production in Bacillus subtilis by Increasing the S-Adenosylmethionine Pool Size

Certain Str(r) mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel(+) but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 μg/ml, an antibiotic not produced (<1 μg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering

Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering

The G243D mutation (afsB mutation) in the principal sigma factor σHrdB alters intracellular ppGpp level and antibiotic production in Streptomyces coelicolor A3(2)

Deficient antibiotic production in an afsB mutant, BH5, of Streptomyces coelicolor A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor sigma(HrdB). Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the afsB(+) parent strain. The introduction of certain rifampicin resistance (rif) mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, actII-ORF4 and redD, for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance (tsp) mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the afsB tsp mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the afsB mutant BH5, resulting in a failure to initiate antibiotic production in this strain.