Our understanding of the mechanisms used byIn spite of a stable decline in the incidence of tuberculosis in countries participating in control surveys, there were an estimated 8.8 million new cases and 1.6 million deaths in 2005 (28). The treatment of Mycobacterium tuberculosis infections requires at least 6 months of antimycobacterial therapy with the use of multiple drugs. This long duration of treatment is justified by the poor efficacy of available antibiotics, including the main drugs isoniazid and rifampin, against the dormant M. tuberculosis bacilli (10, 26) that are thought to persist in particular environments such as the granuloma or caseum (6,21). In vitro models that mimic the persistent state have been developed based on nutrient starvation (4, 12), oxygen deprivation (25), and exposure to nitric oxide (23). These models showed that nonreplicative and low metabolic states of the bacteria could be responsible for the poor in vivo response to currently available drugs. The adaptive response of M. tuberculosis during the transition from aerobic growth to stationary phase results in the activation of a "dormancy" regulon (4, 22, 24). The regulon includes genes that are likely to play an essential role in the long-term survival of the bacteria and therefore encode potential targets for the development of sterilizing drugs.The "dormancy" regulon of M. tuberculosis was not previously reported to include genes involved in peptidoglycan metabolism, although changes in the structure of this cell wall polymer are known to be associated with the transition to the stationary phase in other bacteria. In Escherichia coli, the transition is associated with an increase (1.8 to 5%) in the content of 333 cross-links to the detriment of the classical 433 crosslinks formed by the D,D-transpeptidase activity of penicillinbinding proteins (PBPs) (Fig. 1) (11). We have previously identified the L,D-transpeptidases (Ldt) that catalyze the formation of 333 peptidoglycan cross-links as members of a novel family of active-site cysteine peptidases that have various cellular functions (5,14,15,18). In E. coli, these functions include the anchoring of a lipoprotein to the peptidoglycan in addition to the formation of 333 cross-links (14,15). In a mutant of Enterococcus faecium, an L,D-transpeptidase (Ldt fm ) is the key enzyme of an adaptive response to ␤-lactam antibiotics since it bypasses the D,D-transpeptidase activity of PBPs, leading to high-level resistance to the drugs (18,20).Examination of the microarray data published by Betts et al. showed that an M. tuberculosis gene encoding a member of the active-site cysteine peptidase family, referred to as Rv0116c, of unknown function, was upregulated 17-fold under nutrient starvation (4). We have therefore investigated here the structure of peptidoglycan from M. tuberculosis to evaluate whether formation of 333 peptidoglycan cross-links could be part of