The stringent response of Mycobacterium tuberculosis is required for long-term survival

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-t erm survival under starvation conditions. In gram-negative bacteria, (p)ppG pp is produced by the activity of the related RelA and SpoT proteins. Mycob acterium tuberculosis contains a single homolog of these proteins (Rel(Mtb) ) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) kno ckout strain was constructed in a virulent strain of M. tuberculosis, H37Rv , by allelic replacement. The rel(Mtb) mutant displayed a significantly slo wer aerobic growth rate than the wild type in synthetic liquid media, wheth er rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a hu man macrophage-like cell line. These results suggest that the in vivo carbo n source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-m ediated adaptation. Long-term survival of the rel(Mtb) mutant during in vit ro starvation or nutrient run out in normal media was significantly impaire d compared to that in the wild type. In addition, the mutant was significan tly less able to survive extended anerobic incubation than the wild-type vi rulent organism. Thus, the Rel(Mtb) protein is required for long-term survi val of pathogenic mycobacteria under starvation conditions.