The role of histone-like protein, Hlp, in Mycobacterium smegmatis dormancy.

The role of histone-like protein (Hlp) in the development of a dormant state in long-incubated stationary-phase Mycobacterium smegmatis cells was studied in two models: (1) adoption of 'nonculturable' (NC) state, which is reversible due to resuscitation with proteinaceous resuscitation-promoting factor (Rpf) and (2) the formation of morphologically distinct, ovoid resting forms. In the first model, inactivation of the hlp gene resulted in prolongation of culturability of starved cells followed by irreversible nonculturability when mycobacterial cells were unresponsive to resuscitation with Rpf. In the second model, M. smegmatis strain with the inactivated hlp gene was able to form dormant ovoid cells, but they were less resistant to heating and UV radiation than those of wild-type strain. The susceptibility of ovoid cells produced by Delta hlp mutant to these damaging factors was probably due to a less condensed state of DNA, as revealed by fluorescent microscopy and DAPI staining. Evidently, Hlp is essential for cell viability at a later stage of NC dormancy or provides a greater stability of specialized dormant forms.

Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates Its potential to activate human gammadelta T cells but does not lead to competitive antagonism.

The immunological characterization of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), and its methylenediphosphonate analogue, HMB-PCP, is described. With an EC(50) of 0.1-0.2 nM, HMB-PP is significantly more potent in stimulating human Vgamma9/Vdelta2 T cells than any other compound described so far. However, replacing the pyrophosphate by a P-CH(2)-P function abrogates the bioactivity drastically, with HMB-PCP having a EC(50) of only 5.3 microM.

Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis.

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C. The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.