Summary Languishing antibiotic discovery and flourishing antibiotic resistance have prompted development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against M. tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally-encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2 catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for lassomycin's bacteriocidal activity.

Mycobacteria exhibit two DNA damage response pathways: the LexA/RecA-dependent SOS response and a LexA/RecA-independent pathway. Using a combination of transcriptomics and genome-wide binding site analysis, we demonstrate that PafBC (proteasome accessory factor B and C), encoded in the Pup-proteasome system (PPS) gene locus, is the transcriptional regulator of the predominant LexA/RecA-independent pathway. Comparison of the resulting PafBC regulon with the DNA damage response of Mycobacterium smegmatis reveals that the majority of induced DNA repair genes are upregulated by PafBC. We further demonstrate that RecA, a member of the PafBC regulon and principal regulator of the SOS response, is degraded by the PPS when DNA damage stress has been overcome. Our results suggest a model for the regulation of the mycobacterial DNA damage response that employs the concerted action of PafBC as master transcriptional activator and the PPS for removal of DNA repair proteins to maintain a temporally controlled stress response.

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/ RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/ RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens and represents a major global health problem. Mtb features a multitude of mechanisms that allow the phagocytozed bacteria to withstand the host's innate and adaptive immune response and enable them to persist inside the host macrophages 1,2 . After successful immune evasion the pathogen can persist in the human for decades.A hallmark of activated macrophages is the production of reactive oxygen species and nitric oxide, which damage bacterial proteins, lipids and nucleic acids 3,4 . Thus, in order to maintain the integrity of its genome, Mtb relies on robust mechanisms of DNA damage repair. Whereas in most bacteria the genes involved in these processes are regulated as part of the so-called SOS response, mycobacteria were shown to make use of an additional, independent regulatory mechanism 5-8 . In the SOS response pathway the key regulators are LexA and RecA. Under standard growth conditions the repressor LexA prevents transcription of DNA repair genes by binding to a specific sequence within their promoter, the SOS box 9 . RecA senses DNA damage by binding to single-strand DNA that is generated as a result, thereby forming nucleoprotein filaments. This in turn activates RecA to promote auto-catalytic cleavage of the repressor LexA 9 . As a consequence LexA levels initially decrease and transcription of DNA damage repair genes is de-repressed 10,11 . With only few exceptions, Mtb harbours the genes found in Escherichia coli to mediate a fully functional SOS response 12,13 .Early studies demonstrated that the Mtb recA...

Proteasomal protein degradation exists in mycobacteria and other actinobacteria, and expands their repertoire of compartmentalizing protein degradation pathways beyond the usual bacterial types. A product of horizontal gene transfer, bacterial proteasomes have evolved to support the organism's survival under challenging environmental conditions like nutrient starvation and physical or chemical stresses. Like the eukaryotic 20S proteasome, the bacterial core particle is gated and must associate with a regulator complex to form a fully active protease capable of recruiting and internalizing substrate proteins. By association with diverse regulator complexes that employ different recruitment strategies, the bacterial 20S core particle is able to act in different cellular degradation pathways. In association with the mycobacterial proteasomal ATPase Mpa, the proteasome degrades substrates post-translationally modified with prokaryotic, ubiquitin-like protein Pup in a process called pupylation. Upon interaction with the ATP-independent bacterial proteasome activator Bpa, poorly structured substrates are recruited for proteasomal degradation. A potential third degradation route might employ a Cdc48-like protein of actinobacteria (Cpa), for which interaction with the 20S core was recently demonstrated but no degradation substrates have been identified yet. The alternative interaction partners and wide range of substrate proteins suggest that the bacterial proteasome is a modular, functionally flexible and conditionally regulated degradation machine in bacteria that encounter rapidly changing and challenging conditions.