The hallmark of Mycobacterium tuberculosis is its ability to persist for a long-term in host granulomas, in a non-replicating and drug-tolerant state, and later awaken to cause disease. To date, the cellular factors and the molecular mechanisms that mediate entry into the persistence phase are poorly understood. Remarkably, M. tuberculosis possesses a very high number of toxin-antitoxin (TA) systems in its chromosome, 79 in total, regrouping both well-known (68) and novel (11) families, with some of them being strongly induced in drug-tolerant persisters. In agreement with the capacity of stress-responsive TA systems to generate persisters in other bacteria, it has been proposed that activation of TA systems in M. tuberculosis could contribute to its pathogenesis. Herein, we review the current knowledge on the multiple TA families present in this bacterium, their mechanism, and their potential role in physiology and virulence.

Activators of bacterial σ 54 -RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF , bound to an ATP transition state analog (ADP.AlF x ), in complex with its basal factor σ 54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF at 1.75 Å into the EM map we identify two loops involved in binding σ 54 . By comparing enhancerbinding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ 54 .Gene expression is regulated at the level of RNA polymerase (RNAP) activity. Bacterial RNAP containing the σ 54 factor requires specialized activator proteins, referred to as bacterial Enhancer-Binding Proteins (EBPs) that interact with the basal transcription complex from remote DNA sites by DNA looping (1-4). EBPs bind Upstream Activating Sequences (UAS) via their C-terminal DNA-binding domains and form higher order oligomers that use ATP-hydrolysis to activate transcription (5, 6). EBPs' central σ 54 -RNAP interacting domain is responsible for ATPase activity and transcription activation (7-9) and belongs to the larger AAA+ (ATPase Associated with various cellular Activities) family of proteins (10-12). Well studied EBPs include Phage Shock protein F (PspF), nitrogen fixation protein A (NifA), nitrogen regulation protein C (NtrC), and C 4 -dicarboxylic acid transport protein D (DctD) (1-3, 7, 13).PspF from Escherichia coli forms a stable oligomeric complex with σ 54 at the point of ATP hydrolysis (14). PspF-ADP.AlF x alters the interaction between σ 54 and promoter DNA similarly to PspF hydrolyzing ATP (15), and was thus deemed a functional hydrolysis intermediate. Activator nucleotide-hydrolysis dependent events couple the chemical energy of hydrolysis to transcriptional activation. The highly conserved and EBP-specific GAFTGA amino acid motif (Fig. S1) is a crucial mechanical determinant for the successful transfer of energy from ATP hydrolysis in EBP to the RNAP holoenzyme via σ 54 's small N-terminal * To whom correspondence should be addressed. xiaodong.zhang@imperial.ac.uk. The lack of structural information has hindered progress towards understanding the basis of this energy transfer process required for transcriptional activation. We now present a structure-function analysis of one such system using: 1) a cryo-electron microscopy reconstruction of PspF's AAA+ domain (residues 1-275, PspF ) in complex with σ 54 at the point of ATP hydrolysis (mimicked by in-situ formed ADP.AlF x ), 2) the crystal structure of apo PspF (1-275) at 1.75 Å resolution, and 3) mutational analysis. Europe PMC Funders GroupNano-electro spray mass spectroscopy of a PspF (1-275) -σ 54 complex with ADP.AlF x established that six monomers of PspF are in complex with a monomeric σ 54 , consistent with AAA+ proteins functioning as hexamers (10, 12).The 3-dimensional reconstruction of the...