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.

I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-,ybutyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptinresistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produce submerged spores was largely restored by the addition of decoyinine. This was accompained by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).In bacteria, differentiation and induction of secondary metabolism start concomitantly in response to nutrient limitation. The study of differentiation in Streptomyces spp. has been especially of interest because this genus produces numerous antibiotics by secondary metabolism. The coupling of antibiotic production to morphological differentiation suggests a mechanistic connection which has not been demonstrated. The two processes do, however, often occur in parallel, (5). Typically, Streptomyces spp. develop aerial mycelia from substrate mycelia prior to the formation of spores (aerial sp...

Genome sequencing of Streptomyces, myxobacteria, and fungi showed that although each strain contains genes that encode the enzymes to synthesize a plethora of potential secondary metabolites, only a fraction are expressed during fermentation. Interest has therefore grown in the activation of these cryptic pathways. We review current progress on this topic, describing concepts for activating silent genes, utilization of “natural” mutant-type RNA polymerases and rare earth elements, and the applicability of ribosome engineering to myxobacteria and fungi, the microbial groups known as excellent searching sources, as well as actinomycetes, for secondary metabolites.

Guanosine-tetraphosphate (ppGpp) is a major regulator of stringent control, an adaptive response of bacteria to amino acid starvation. The 2.7 A resolution structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to the same site near the active center in both independent RNAP molecules in the crystal but in strikingly distinct orientations. Binding is symmetrical with respect to the two diphosphates of ppGpp and is relaxed with respect to the orientation of the nucleotide base. Different modes of ppGpp binding are coupled with asymmetry of the active site configurations. The results suggest that base pairing of ppGpp with cytosines in the nontemplate DNA strand might be an essential component of transcription control by ppGpp. We present experimental evidence highlighting the importance of base-specific contacts between ppGpp and specific cytosine residues during both transcription initiation and elongation.

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys883Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin-or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.