We report on a target-based approach to identify possible Mycobacterium tuberculosis DXS inhibitors from the structure of a known transketolase inhibitor. A small focused library of analogs was assembled in order to begin elucidating some meaningful structure–activity relationships of 3-(4-chloro-phenyl)-5-benzyl-4H-pyrazolo[1,5-a]pyrimidin-7-one. Ultimately we found that 2-methyl-3-(4-fluorophenyl)-5-(4-meth-oxy-phenyl)-4H-pyrazolo[1,5-a]pyrimidin-7-one, although still weak, was able to inhibit M. tuberculosis DXS with an IC50 of 10.6 μM.
Drug design; DXS; Enzyme; SAR; Tuberculosis
MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.
A concise total synthesis of capuramycin (1), a promising preclinical TB drug lead, is achieved by high-yield formations of the cyanohydrin 5a and 4″,5″-glycal derivative 12. Capuramycin can be synthesized in eight steps from the uridine building block 5a with >30% overall yield. The synthetic intermediates reported here are useful for generation of analogs to improve pharmacokinetic properties of capuramycin.
Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.
Malaria; Menaquinone; Apicomplexa; Ubiquinone; Vitamin K; Plasmodium falciparum
cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.
Mycobacterium tuberculosis; Isoprenoid; Prenyltransferase; Prenyl diphosphate synthase; Undecaprenyl diphosphate synthase; Chain-length determination mechanism; Mutagenesis
With the increased need for novel antimicrobials to improve the existing treatment for tuberculosis, to combat multidrug-resistant tuberculosis, and to address the presence of latent bacilli in a large population throughout the world, which can reactivate and cause active disease, there is a need for rapid, low-cost, high-throughput assays for screening new drug candidates. A micro-plate-based Alamar blue assay meets these requirements. In addition to the identification of the antimicrobial activities of compounds, determination of their toxicities is important. The high costs involved in testing compounds in whole animal models has led to the development of in vitro cytotoxicity assays using human and animal cell lines. Microplate-based Alamar blue and cytotoxicity assays have been applied to search for novel antimicrobials to treat tuberculosis. These methods are described in detail herein.
Alamar blue; cytotoxicity; MIC; Mycobacterium tuberculosis; tetrazolium
The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.
Mycobacteria have a unique cell wall, which is rich in drug targets. The cell wall core consists of a peptidoglycan layer, a mycolic acid layer, and an arabinogalactan polysaccharide connecting them. The detailed structure of the cell wall core is largely, although not completely, understood and will be presented. The biosynthetic pathways of all three components reveal significant drug targets that are the basis of present drugs and/or have potential for new drugs. These pathways will be reviewed and include enzymes involved in polyisoprene biosynthesis, soluble arabinogalactan precursor production, arabinogalactan polymerization, fatty acid synthesis, mycolate maturation, and soluble peptidoglycan precursor formation. Information relevant to targeting all these enzymes will be presented in tabular form. Selected enzymes will then be discussed in more detail. It is thus hoped this chapter will aid in the selection of targets for new drugs to combat tuberculosis.
Tuberculosis; drug discovery; cell wall; arabinogalactan; mycolic acids; peptidoglycan; drug target
Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low μM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labeling studies and quantitation of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.
Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogs of the new tuberculosis (TB) drug SQ109 (1) which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multi-target inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.
Menaquinone (MK) with partially saturated
isoprenyl moieties is
found in a wide range of eubacteria and Archaea. In many Gram-positive
organisms, including mycobacteria, it is the double bond found in
the β-isoprene unit that is reduced. Mass spectral characterization
of menaquinone from mycobacterial knockout strains and heterologous
expression hosts demonstrates that Rv0561c (designated menJ) encodes an enzyme which reduces the β-isoprene
unit of menaquinone in Mycobacterium tuberculosis, forming the predominant form of menaquinone found in mycobacteria.
MenJ is highly conserved in mycobacteria species but is not required
for growth in culture. Disruption of menJ reduces
mycobacterial electron transport efficiency by 3-fold, but mycobacteria
are able to maintain ATP levels by increasing the levels of the total
menaquinone in the membrane; however, MenJ is required for M. tuberculosis survival in host macrophages. Thus, MK with
partially hydrogenated isoprenyl moieties represents a novel virulence
factor and MenJ is a contextually essential enzyme and a potential
drug target in pathogenic mycobacteria and other Gram-positive pathogens.
Rv0561c (MenJ) reduces the β-isoprene
unit of menaquinone
in Mycobacterium tuberculosis, which increases electron
transport efficiency and is required for bacterial survival in macrophages.
Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-14C]cholesterol or [26-14C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.
IMPORTANCE Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.
Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.
This chapter summarizes what is currently known of the structures, physiological roles, involvement in pathogenicity and biogenesis of a variety of non-covalently bound cell envelope lipids and glycoconjugates of Mycobacterium tuberculosis and other Mycobacterium species. Topics addressed in this chapter include phospholipids; phosphatidylinositol mannosides; triglycerides; isoprenoids and related compounds (polyprenyl phosphate, menaquinones, carotenoids, non-carotenoid cyclic isoprenoids); acyltrehaloses (lipooligosaccharides, trehalose mono- and di-mycolates, sulfolipids, di- and poly-acyltrehaloses); mannosyl-beta-1-phosphomycoketides; glycopeptidolipids; phthiocerol dimycocerosates, para-hydroxybenzoic acids and phenolic glycolipids; mycobactins; mycolactones; and capsular polysaccharides.
Analytical probes capable of mapping molecular composition at the nanoscale are of critical importance to materials research, biology and medicine. Mass spectral imaging makes it possible to visualize the spatial organization of multiple molecular components at a sample's surface. However, it is challenging for mass spectral imaging to map molecular composition in three dimensions (3D) with submicron resolution. Here we describe a mass spectral imaging method that exploits the high 3D localization of absorbed extreme ultraviolet laser light and its fundamentally distinct interaction with matter to determine molecular composition from a volume as small as 50 zl in a single laser shot. Molecular imaging with a lateral resolution of 75 nm and a depth resolution of 20 nm is demonstrated. These results open opportunities to visualize chemical composition and chemical changes in 3D at the nanoscale.
Mass spectral analysis is used to map the composition of materials and surfaces in numerous fields. Here, the authors report a mass spectral technique based on extreme ultraviolet laser ablation that allows three-dimensional imaging of chemical composition in addition to giving highly sensitive nanoscale resolution.
We report the discovery of a series
of new drug leads that have
potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite.
The compounds are analogues of the new tuberculosis (TB) drug SQ109
(1), which has been reported to act by inhibiting a transporter
called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone
biosynthesis and electron transport, inhibiting respiration and ATP
biosynthesis, and are uncouplers, collapsing the pH gradient and membrane
potential used to power transporters. The result of such multitarget
inhibition is potent inhibition of TB cell growth, as well as very
low rates of spontaneous drug resistance. Several targets are absent
in humans but are present in other bacteria, as well as in malaria
parasites, whose growth is also inhibited.
have obtained the structure of the bacterial diterpene synthase,
tuberculosinol/iso-tuberculosinol synthase (Rv3378c)
from Mycobacterium tuberculosis, a
target for anti-infective therapies that block virulence factor formation.
This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures
containing the tuberculosinyl diphosphate substrate together with
one bisphosphonate inhibitor-bound structure. These structures together
with the results of site-directed mutagenesis suggest an unusual mechanism
of action involving two Tyr residues. Given the similarity in local
and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c),
the possibility exists for the development of inhibitors that target
not only virulence but also cell wall biosynthesis, based in part
on the structures reported here.
Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.
Aurachin RE (1) is a strong antibiotic that was recently found to possess MenA (1,4-dihydroxy-2-naphthoate prenyltransferase) and bacterial electron transport inhibitory activities. Aurachin RE is the only molecule in a series of aurachin natural products that has the chiral center in the alkyl side chain at C9′-position. To identify selective MenA inhibitors against Mycobacterium tuberculosis, a series of chiral molecules were designed based on the structures of previously identified MenA inhibitors and 1. The synthesized molecules were evaluated in in vitro assays including MenA enzyme and bacterial growth inhibitory assays. We could identify novel MenA inhibitors that showed significant increase in potency of killing non-replicating M. tuberculosis in the low oxygen recovery assay (LORA) without inhibiting other Gram-positive bacterial growth even at high concentrations. The MenA inhibitors reported here are useful new pharmacophores for the development of selective antimycobacterial agents with strong activity against non-replicating M. tuberculosis.
New antimycobacterial agent; Menaquinone biosynthesis inhibitor; MDR Mycobacterium tuberculosis; Non-replicating Mycobacterium tuberculosis; TB drugs; quinolone alkaloid; aurachin RE
1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a novel target for developing new antibacterial (including anti-tuberculosis) and antimalaria drugs. 41 lipophilic phosphonates, representing a new class of DXR inhibitors, were synthesized, among which 5-phenylpyridin-2-ylmethylphosphonic acid possesses the most activity against E. coli DXR (EcDXR) with a Ki of 420 nM. Structure activity relationships (SAR) are discussed, which can be rationalized using our EcDXR:inhibitor structures, and a predictive quantitative SAR (QSAR) model is also developed. Since inhibition studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been well performed, 48 EcDXR inhibitors with a broad chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a MtDXR:inhibitor complex reveals the flexible loop containing the residues 198–208 has no strong interactions with the 3,4-dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide implications in the future design and development of potent DXR inhibitors.
Lipoarabinomannan (LAM) is a structurally heterogeneous amphipathic lipoglycan present in Mycobacterium spp. and other actinomycetes, which constitutes a major component of the cell wall and exhibits a wide spectrum of immunomodulatory effects. Analysis of Mycobacterium smegmatis subcellular fractions and spheroplasts showed that LAM and lipomannan (LM) were primarily found in a cell wall-enriched subcellular fraction and correlated with the presence (or absence) of the mycolic acids in spheroplast preparations, suggesting that LAM and LM are primarily associated with the putative outer membrane of mycobacteria. During the course of these studies significant changes in the LAM/LM content of the cell wall were noted relative to the age of the culture. The LAM content of the M. smegmatis cell wall was dramatically reduced as the bacilli approached stationary phase, whereas LM, mycolic acid, and arabinogalactan content appeared to be unchanged. In addition, cell morphology and acid-fast staining characteristics showed variations with growth phase of the bacteria. In the logarithmic phase, the bacteria were found to be classic rod-shaped acid-fast bacilli, while in the stationary phase M. smegmatis lost the characteristic rod shape and developed a punctate acid-fast staining pattern with carbolfuchsin. The number of viable bacteria was independent of LAM content and phenotype. Taken together, the results presented here suggest that LAM is primarily localized with the mycolic acids in the cell wall and that the cellular concentration of LAM in M. smegmatis is selectively modulated with the growth phase.
Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyl-transferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac2PIM2), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC–liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose linear α-d-Araf-(1→5)3–5 oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.
The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.