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-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether 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 human macrophage-like cell line. These results suggest that the in vivo carbon 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)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.     

Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models

Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. The stem cell undergoes self-renewal throughout life, but study of its biological characteristics has been difficult because a very small number (2 to 3 in 10(4) cells) exist in the testis and they can only be identified by function. Although the development of the spermatogonial transplantation technique has provided an assay system for stem cells, efficient methods to enrich stem cells have not been available. Here, we examined two infertile mouse models, Steel/Steel(Dickie)(Sl/Sl(d)) and experimental cryptorchid, as a source of testis cell populations enriched in stem cells. The Sl/Sl(d) testis showed little enrichment, which raises questions about how adult stem cell number is determined and about the currently accepted belief that adult stem cells are independent of Sl factor. The cells recovered from cryptorchid testes were enriched for stem cells 25-fold (colonies) or 50-fold (area) compared to wild-type testes. The cryptorchid condition does not affect stem cell activity, but eliminates almost all differentiated cells, and about 1 in 200 cells is a stem cell. Thus, cryptorchid testes provide an important approach for purification and characterization of spermatogonial stem cells.     

Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis.

A 2.2kb relA/spoT homologue was isolated from Mycobacterium tuberculosis (Mtb) genomic DNA by PCR-amplification. The Mtb gene encodes a protein of 738 amino acid residues, and is flanked upstream by an ORF that is highly similar to the apt gene, and downstream by an ORF that is highly similar to the cypH gene. This dual function Mtb homologue belongs to the relA/spoT family of genes that mediate the stringent response by regulating the synthesis and degradation of guanosine 3',5'-bis(diphosphate) (ppGpp) and pppGpp. In vitro biochemical data indicate that purified RelMtb is a ribosome- and tRNA-independent ATP:GTP/GDP/ITP 3'-pyrophosphoryltransferase. Additionally, purified RelMtb is an Mn2+-dependent, ribosome and tRNA-independent, (p)ppGpp 3'-pyrophosphohydrolase. These reactions were also assessed in vivo in E. coli deleted in both the relA and spoT genes, which generates a (p)ppGpp0 phenotype. RelMtb can suppress this phenotype and can generate more (p)ppGpp than relA in the wild type E. coli control.