From microbial differentiation to ribosome engineering.

Bacillus subtilis and Streptomyces spp. provide tractable experimental systems for studying cellular responses to adverse environmental conditions. During conditions of extreme nutrient limitation, these prokaryotes exhibit a wide range of adaptations, including the production and secretion of antibiotics and enzymes and the formation of aerial mycelium and spores. In response to these conditions, all bacteria, but not eukaryotic microorganisms, exhibit a "stringent response," during which the unusual guanosine tetraphosphate, ppGpp, accumulates intracellularly. This is accompanied by a marked reduction in the GTP pool, due to ppGpp inhibition of IMP-dehydrogenase, and immediate repression of rRNA synthesis, due to the binding of ppGpp to RNA polymerase. This review summarizes our studies on the bacterial stringent response and its use in applied microbiology. We found that morphological differentiation results from a decrease in the pool of GTP, whereas physiological differentiation (antibiotic production) results from a more direct function of ppGpp. That is, we found that the Streptomyces GTP-binding protein Obg functions by sensing intracellular GTP levels and that certain mutations in the RNA polymerase beta-subunit circumvent dependence on ppGpp in antibiotic production. X-ray crystallographic analysis provided a structural basis for the ppGpp regulation of transcription. On the basis of these findings, we have developed the novel concept of "ribosome engineering," focusing on activation of dormant genes to elicit cellular function fully. Ribosome engineering can be applied to strain improvement, screening of novel metabolites, plant breeding, cell-free translation systems, and the treatment of tuberculosis.