The growth-regulating property of M. tuberculosis Rv2623 is dependent on its ability to bind ATP 
We next sought to probe the relationship between the nucleotide-binding capacity and growth regulation by this mycobacterial USP.
Both the D15E and G117A mutant proteins were overexpressed in M. smegmatis mc2155 at levels equivalent to that of wild-type Rv2623 (Figure S4).
Results of these studies demonstrated that while overexpression of wildtype Rv2623 retards the growth of the recipient strain relative to cells transformed with vector alone, growth of the strains overexpressing ATP-binding-deficient mutant Rv2623 are only minimally affected by overexpression as assessed by spotting serial dilutions of the cultures of the appropriate strains onto solid Middlebrook 7H10 agar (data not shown) as well as by monitoring the time to detection using the BD BACTEC 9000MB system (Figure 8A).
The distinct effects exhibited by the wild type and the G117A and D15E mutants defective in ATP binding suggests a direct correlation between growth attenuation and ATP binding (Figure 7B).
To examine whether the effects of overexpression of Rv2623 on M. smegmatis are operative in virulent M. tuberculosis, the growth kinetics of the Erdman strain overexpressing wildtype Rv2623, as well as the Rv2623G117A and the Rv2623D15E mutant proteins, were evaluated in vitro using the BACTEC 9000MB system (Figure 8B).
As in the M. smegmatis studies, the results show that overexpression of Rv2623 in M. tuberculosis results in marked retardation of growth.
Furthermore, this growth attenuation is not observed in M. tuberculosis strains overexpressing the G117A or the D15E mutant Rv2623 (Figure 8B).
Taken together, these data strongly suggest that the ability of Rv2623 to regulate growth of M. smegmatis and M. tuberculosis is dependent on an ATP-dependent process.
