Results 
Mycobacterium tuberculosis Rv2623 regulates growth in vitro An rv2623-deletion mutant of the virulent M. tuberculosis Erdman strain was generated by specialized transduction [12].
The rv2623-specific allelic exchange construct was delivered via recombinant mycobacteriophage phAE159 and transformants were analyzed by Southern blot, confirming replacement of rv2623 with the hyg gene, which confers hygromycin resistance (Figure 1A).
Aliquots of a single knockout clone, designated as Deltarv2623, were stored at -70degreesC.
Deletion of rv2623 is not likely to affect transcription of neighboring genes, given the sequence-confirmed precise excision of the rv2623 coding region and the gene organization at the rv2623 locus (the downstream rv2624c is transcribed in the direction opposite to that of rv2623) (Figure 1B).
Deletion of specific USPs in E. coli results in growth defects in vitro [8],[13],[14].
For example, an E. coli strain deficient for UspA exhibits reduced survival in stationary phase culture [14].
However, the in vitro growth kinetics of Deltarv2623 M. tuberculosis in OADC-supplemented Middlebrook 7H9 or minimal Sauton's medium is comparable to that of wildtype Erdman up to 14 days post-inoculation (Figure 2A).
We reasoned that a potential growth-regulating attribute of Rv2623 might be masked by functional redundancy among the M. tuberculosis USP homologs.
Indeed, partial functional overlap has been demonstrated among the E. coli USPs [9],[15].
We therefore examined the effect of overexpression of this USP in the rapidly growing M. smegmatis strain mc2155 [16].
As seen in Figure 2B, constitutive overexpression of M. tuberculosis rv2623 using the multi-copy plasmid pMV261 resulted in growth deficiency of the recipient strain both on solid medium (Middlebrook 7H10 agar) and in the liquid medium-based BD BACTEC 9000MB system.
These results strongly suggest that M. tuberculosis Rv2623 regulates mycobacterial growth in vitro.
