DUF2285 is a novel helix-turn-helix domain variant that orchestrates both activation and antiactivation of conjugative element transfer in proteobacteria.

Horizontal gene transfer is tightly regulated in bacteria. Often only a fraction of cells become donors even when regulation of horizontal transfer is coordinated at the cell population level by quorum sensing. Here, we reveal the widespread 'domain of unknown function' DUF2285 represents an 'extended-turn' variant of the helix-turn-helix domain that participates in both transcriptional activation and antiactivation to initiate or inhibit horizontal gene transfer. Transfer of the integrative and conjugative element ICEMlSymR7A is controlled by the DUF2285-containing transcriptional activator FseA. One side of the DUF2285 domain of FseA has a positively charged surface which is required for DNA binding, while the opposite side makes critical interdomain contacts with the N-terminal FseA DUF6499 domain. The QseM protein is an antiactivator of FseA and is composed of a DUF2285 domain with a negative surface charge. While QseM lacks the DUF6499 domain, it can bind the FseA DUF6499 domain and prevent transcriptional activation by FseA. DUF2285-domain proteins are encoded on mobile elements throughout the proteobacteria, suggesting regulation of gene transfer by DUF2285 domains is a widespread phenomenon. These findings provide a striking example of how antagonistic domain paralogues have evolved to provide robust molecular control over the initiation of horizontal gene transfer.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element.

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

Antibiotic resistance in Mycobacterium tuberculosis alters tolerance to cell wall-targeting inhibitors.

Background: A limited ability to eliminate drug-resistant strains of Mycobacterium tuberculosis is a major contributor to the morbidity of TB. Complicating this problem, little is known about how drug resistance-conferring mutations alter the ability of M. tuberculosis to tolerate antibiotic killing. Here, we investigated if drug-resistant strains of M. tuberculosis have an altered ability to tolerate killing by cell wall-targeting inhibitors. Methods: Bacterial killing and MIC assays were used to test for antibiotic tolerance and synergy against a panel of drug-resistant M. tuberculosis strains. Results: Our results demonstrate that vancomycin and thioacetazone exhibit increased killing of diverse drug-resistant strains. Mutations in mmaA4 and mmpL3 increased vancomycin killing, which was consistent with vancomycin synergizing with thioacetazone and MmpL3-targeting inhibitors. In contrast, mutations in the mce1 operon conferred tolerance to vancomycin. Conclusions: Overall, this work demonstrates how drug-resistant strains experience perturbations in cell-wall production that alters their tolerance to killing by cell wall-targeting inhibitors.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

Uncovering interactions between mycobacterial respiratory complexes to target drug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times. Realizing the full potential of this unique target space requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest interactions and potential in a clinical setting. In this review, we discuss (i) chemical-interaction, (ii) genetic-interaction and (iii) chemical-genetic interaction studies to explore the consequences of inhibiting multiple mycobacterial respiratory components. We provide potential mechanisms to describe the basis for the strongest interactions. Finally, whilst we place an emphasis on interactions that occur with existing bioenergetic inhibitors, by highlighting interactions that occur with alternative respiratory components we envision that this information will provide a rational to further explore alternative proteins as potential drug targets and as part of unique drug combinations.