Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

28Farmed ruminants are the largest source of anthropogenic methane emissions 29 globally. The methanogenic archaea responsible for these emissions use molecular 30 hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, 31 as their primary energy source. In this work, we used comparative genomic, 32 metatranscriptomic, and co-culture-based approaches to gain a system-wide 33 understanding of the organisms and pathways responsible for ruminal H2 metabolism. 34 Two thirds of sequenced rumen bacterial and archaeal genomes encode enzymes 35 that catalyze H2 production or consumption, including 26 distinct hydrogenase 36 subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are 37 differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases 38 from carbohydrate-fermenting Clostridia (e.g. Ruminococcus) accounted for half of all 39 hydrogenase transcripts. Various H2 uptake pathways were also expressed, including 40 methanogenesis (Methanobrevibacter), fumarate reduction and nitrate ammonification 41 (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis predominated 42 in high methane yield sheep, alternative uptake pathways were significantly 43 upregulated in low methane yield sheep. Complementing these findings, we observed 44 significant differential expression and activity of the hydrogenases of the 45 hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic 46 fumarate reducer Wolinella succinogenes in co-culture compared to pure culture. We 47 conclude that H2 metabolism is a more complex and widespread trait among rumen 48 microorganisms than previously recognized. There is evidence that alternative 49 hydrogenotrophs, including acetogens and selenomonads, can prosper in the rumen 50 and effectively compete with methanogens for H2 in low methane yield ruminants. 51Strategies to increase flux through alternative H2 uptake pathways, including animal 52 selection, dietary supplementation, and methanogenesis inhibitors, may lead to 53 sustained methane mitigation. 54 55 56Methane production by livestock accounts for over 5% of global greenhouse gas 57 emissions annually 1 . These emissions mostly originate from the activity of 58 methanogens within ruminants, which generate methane as an obligate end-product 59 of their energy metabolism 2 . Several lineages of methanogenic archaea are core 60 members of the microbiome of the ruminant foregut 3-5 . Of these, hydrogenotrophic 61 methanogens are dominant in terms of both methane emissions and community 62 composition 6,7 , with global surveys indicating that Methanobrevibacter gottschalkii 63 and Methanobrevibacter ruminantium comprise 74% of the rumen methanogen 64 community 5 . These organisms use molecular hydrogen (H2) to reduce carbon dioxide 65 (CO2) to methane through the Wolfe cycle of methanogenesis 8,9 . Rumen 66 methanogens have also been identified that use formate, acetate, methyl compounds, 67 and ethanol as substrates, but usually do so ...

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions suggesting that regimens with multiple bioenergetic inhibitors will drastically shorten treatment times. However, realizing this potential is hampered by a lack of on-target inhibitors and a poor understanding of which inhibitor combinations have the strongest interactions. To overcome these limitations, we have used CRISPR interference (CRISPRi) to characterize the consequences of transcriptionally inhibiting individual respiratory complexes and identify bioenergetic complexes that when simultaneously inhibited result in cell death. In this study, we identified known and novel synthetic lethal interactions and demonstrate how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.