SummaryNon-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membraneanchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed nonoverlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

Resistance of Enterococcus faecalis against antimicrobial peptides, both of host origin and produced by other bacteria of the gut microflora, is likely to be an important factor in the bacterium's success as an intestinal commensal. The aim of this study was to identify proteins with a role in resistance against the model antimicrobial peptide bacitracin. Proteome analysis of bacitracintreated and untreated cells showed that bacitracin stress induced the expression of cell wall-biosynthetic proteins and caused metabolic rearrangements. Among the proteins with increased production, an ATP-binding cassette (ABC) transporter with similarity to known peptide antibiotic resistance systems was identified and shown to mediate resistance against bacitracin. Expression of the transporter was dependent on a two-component regulatory system and a second ABC transporter, which were identified by genome analysis. Both resistance and the regulatory pathway could be functionally transferred to Bacillus subtilis, proving the function and sufficiency of these components for bacitracin resistance. Our data therefore show that the two ABC transporters and the two-component system form a resistance network against antimicrobial peptides in E. faecalis, where one transporter acts as the sensor that activates the TCS to induce production of the second transporter, which mediates the actual resistance.

SummaryGenes with a role in proline metabolism are strongly expressed when mycobacterial cells are exposed to nutrient starvation and hypoxia. Here we show that proline metabolism in mycobacteria is mediated by the monofunctional enzymes D 1 -pyrroline-5-carboxylate dehydrogenase (PruA) and proline dehydrogenase (PruB). Proline metabolism was controlled by a unique membrane-associated DNA-binding protein PruC. Under hypoxia, addition of proline led to higher biomass production than in the absence of proline despite excess carbon and nitrogen. To identify the mechanism responsible for this enhanced growth, microarray analysis of wild-type Mycobacterium smegmatis versus pruC mutant was performed. Expression of the DNA repair machinery and glyoxalases was increased in the pruC mutant. Glyoxalases are proposed to degrade methylglyoxal, a toxic metabolite produced by various bacteria due to an imbalance in intermediary metabolism, suggesting the pruC mutant was under methylglyoxal stress. Consistent with this notion, pruB and pruC mutants were hypersensitive to methylglyoxal. D 1 -pyrroline-5-carboxylate is reported to react with methylglyoxal to form non-toxic 2-acetyl-1-pyrroline, thus providing a link between proline metabolism and methylglyoxal detoxification. In support of this mechanism, we show that proline metabolism protects mycobacterial cells from methylglyoxal toxicity and that functional proline dehydrogenase, but not D 1 -pyrroline-5-carboxylate dehydrogenase, is essential for this protective effect.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a highthroughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.KEYWORDS methanogen, greenhouse gas, Methanococcus maripaludis, highthroughput, rumen, Methanobrevibacter M ethane emissions from ruminants are a significant contributor to global greenhouse gas emissions (1). In countries such as New Zealand, with a large pasturebased livestock sector, greenhouse gas emissions from agriculture represent approxi-

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