Structure of an endogenous mycobacterial MCE lipid transporter.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug­drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical­genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo­relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical­genetic­environmental interactions that can be used to optimize drug­drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

Mycobacterial Membranes as Actionable Targets for Lipid-Centric Therapy in Tuberculosis.

Infectious diseases remain significant health concerns worldwide, and resistance is particularly common in patients with tuberculosis caused by Mycobacterium tuberculosis. The development of anti-infectives with novel modes of action may help overcome resistance. In this regard, membrane-active agents, which modulate membrane components essential for the survival of pathogens, present attractive antimicrobial agents. Key advantages of membrane-active compounds include their ability to target slow-growing or dormant bacteria and their favorable pharmacokinetics. Here, we comprehensively review recent advances in the development of membrane-active chemotypes that target mycobacterial membranes and discuss clinically relevant membrane-active antibacterial agents that have shown promise in counteracting bacterial infections. We discuss the relationship between the membrane properties and the synthetic requirements within the chemical scaffold, as well as the limitations of current membrane-active chemotypes. This review will lay the chemical groundwork for the development of membrane-active antituberculosis agents and will foster the discovery of more effective antitubercular agents.

Structure and dynamics of a mycobacterial type VII secretion system.

Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems-known as type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.

Discovery of ANTAR-RNAs and their Mechanism of Action in Mycobacteria.

Non-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) still remain elusive. Here, by re-engineering the existing genome-wide search approach, we discover a family of structurally conserved RNAs that are present ubiquitously across actinobacteria, including mycobacteria. In vitro analysis shows that RNAs belonging to this family bind response-regulator proteins that contain the widely prevalent ANTAR domain. The Mycobacterium tuberculosis ANTAR protein gets phosphorylated by a histidine kinase and interacts with RNA only in its phosphorylated state. These newly identified RNAs reside only in certain transcripts and typically overlap with the ribosome-binding site, regulating translation of these transcripts. In this way, the RNAs directly link signaling pathways to translational control, thus expanding the mechanistic tool kit available for ANTAR-based control of gene expression. In mycobacteria, we find that RNAs targeted by ANTAR proteins majorly encode enzymes of lipid metabolism and associated redox pathways. This now allows us to identify the key genes that mediate ANTAR-dependent control of lipid metabolism. Our study establishes the identity and wide prevalence of ANTAR-target RNAs in mycobacteria, bringing RNA-mediated regulation in these bacteria to the center stage.

Interaction of 5-substituted pyrimidine nucleoside analogues and M.Tuberculosis: A view through an electron microscope.

The data of transmission electron microscopy (TEM) on morphology of M. tuberculosis H37Rv bacterial cells treated with four analogues of pyrimidine nucleosides with different substituents at 5 position of base are presented. We showed that the growth of M. tuberculosis H37Rv cells effectively inhibited by each of these compounds. This process is accompanied with the accumulation of lipid intracellular vacuole-like inclusions in the cells, appearance of deep protrusions and indentations on the surface, partial and/or complete destruction of the three-layered cell envelope. The exact molecular mechanism of action of 5-substituted pyrimidine nucleosides on M. tuberculosis cells remains to be proved. However, one can suggest that mechanism of action for these compounds is related either to their direct interactions with bacteria cell walls or to interactions with enzymes participating in the process of cell wall formation.

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis.

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It consists of five α-helices, whereas all other HLP domains have four. In contrast with other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably, a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five α-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in the Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for the interaction of Mka HLP irons with nitric oxide.

Potential Drug Targets in Mycobacterial Cell Wall: Non-Lipid Perspective.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), still remains a deadly disease worldwide. With prolonged usage of anti-TB drugs, the current therapeutic regimes are becoming ineffective, particularly due to emergence of drug resistance in MTB. Under such compelling circumstances, it is pertinent to look for new drug targets. The cell wall envelope of MTB is composed of unique lipids that are frequently targeted for anti-TB therapy. This is evident from the fact that most of the commonly used front line drugs (Isoniazid and Ethambutol) act on lipid machinery of MTB. Thus, despite the fact that much of the attention is towards understanding the MTB lipid biology, in search for identification of new drug targets, our knowledge of bacterial cell wall non-lipid components remains rudimentary and underappreciated. Better understanding of such components of mycobacterial cell structure will help in the identification of new drug targets that can be utilized on the persistent mycobacterium. This review at a common platform summarizes some of the non-lipid cell wall components in MTB that have potential to be exploited as future drug targets.

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31.

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.

The ATPases of the mycobacterial type VII secretion system: Structural and mechanistic insights into secretion.

Tuberculosis (TB) remains the foremost cause of death by infectious disease and is propagated by the pathogen Mycobacterium tuberculosis (Mtb). The virulence associated with Mtb is mediated by proteins secreted into host cells by the type VII secretion system (T7SS), making this system a candidate for future drug and vaccine development. However, while many of the components involved in the T7SS have been identified, the mechanism of translocation across both the inner and outer mycobacterial membranes remains largely unexplained. Key to the translocation of proteins across the membrane is the activity of conserved AAA+ ATPases EccA and EccC, which are explored in this review. Although the T7SS does not appear homologous to other known bacterial secretion systems, many of those require ATPase activity during different phases of protein translocation. Thus, exploring the roles of ATPases in multiple secretion systems may provide insights into the T7SS. Targeting bacterial virulence factors such as secretion systems is becoming an increasingly explored area of research, and here we review how such strategies could be applied to the T7SS.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target.

Despite intensive efforts to discover highly effective treatments to eradicate tuberculosis (TB), it remains as a major threat to global human health. For this reason, new TB drugs directed toward new targets are highly coveted. MmpLs (Mycobacterial membrane proteins Large), which play crucial roles in transporting lipids, polymers and immunomodulators and which also extrude therapeutic drugs, are among the most important therapeutic drug targets to emerge in recent times. Here, crystal structures of mycobacterial MmpL3 alone and in complex with four TB drug candidates, including SQ109 (in Phase 2b-3 clinical trials), are reported. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear to be key facilitators of proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs. This structural data will greatly advance the development of MmpL3 inhibitors as new TB drugs.

Retention of EsxA in the Capsule-Like Layer of Mycobacterium tuberculosis Is Associated with Cytotoxicity and Is Counteracted by Lung Surfactant.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cell's bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome.

Ultrastructural analysis of cell wall of drug resistant and sensitive Mycobacterium tuberculosis isolated from cerebrospinal fluid by transmission electron microscope.

Drug-resistant tuberculosis is being increasingly recognized and is one among the leading cause of death worldwide. Remarkable impermeability of cell wall to antituberculous drugs protects the mycobacteria from drug action. The present study analyzed the cell wall thickness among first-line drug resistant and sensitive Mycobacterium tuberculosis (Mtb) isolated from cerebrospinal fluid by transmission electron microscopy (TEM). The average thickness of the cell wall of sensitive isolates was 13.60 ± 0.98 nm. The maximum difference (26.48%) in the cell wall thickness was seen among multi-drug resistant (18.50 ± 1.71 nm) isolates and the least difference (4.14%) was shown by streptomycin-resistant (14.18 ± 1.38 nm) isolates. The ultrastructural study showed evident differences in the cell wall thickness among sensitive and resistant isolates. Preliminary TEM examination of cells indicates that morphological changes occur in the cell wall which might be attributed to the drug resistance. The thickened wall of Mtb appears to help the bacilli to overcome the action of antituberculous drugs.

Novel 5'-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents.

A series of novel 5'-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 µg/mL (mc²155) and a MIC99 of 6.7⁻67 µg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28⁻61 µg/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50⁻60 µg/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC99 20⁻50 µg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20⁻50 µg/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Structure and function of Mycobacterium-specific components of F-ATP synthase subunits α and ε.

The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system αchi3:ß3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:ß3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:ß3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.

Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis.

In this study, the electropolymerization of 4-hydroxyphenylacetic acid (4-HPA) over graphite electrodes (GE) was optimized, aiming its application as a functionalized electrochemical platform for oligonucleotides immobilization. It was investigated for the number of potential cycles and the scan rate influence on the monomer electropolymerization by using cyclic voltammetry technique. It was observed that the polymeric film showed a redox response in the region of +0.53/+0.38 V and the increase in the number of cycles produces more electroactive platforms because of the better electrode coverage. On the other hand, the decrease of scan rate produces more electroactive platforms because of the occurrence of more organized coupling. Scanning electron microscopy (SEM) images showed that the number of potential cycles influences the coverage and morphology of the electrodeposited polymeric film. However, the images also showed that at different scan rates a more organized material was produced. The influence of these optimized polymerization parameters was evaluated both in the immobilization of specific oligonucleotides and in the detection of hybridization with complementary target. Poly(4-HPA)/GE platform has shown efficient and sensitive for oligonucleotides immobilization, as well as for a hybridization event with the complementary oligonucleotide in all investigated cases. The electrode was modified with 100 cycles at 75 mV/s presented the best responses in function of the amplitude at the monitored peak current values for the Methylene Blue and Ethidium Bromide intercalators. The construction of the genosensor to detect a specific oligonucleotide sequence for the Mycobacterium tuberculosis bacillus confirmed the results regarding the poly(4-HPA)/GE platform efficiency since it showed excellent sensitivity. The limit of detection and the limit of quantification was found to be 0.56 (±0.05) µM and 8.6 (±0.7) µM, respectively operating with very low solution volumes (15 µL of probe and 10 µL target). The biosensor development was possible with optimization of the probe adsorption parameters and target hybridization, which led to an improvement in the decrease of the Methylene Blue (MB) reduction signal from 14% to 34%. In addition, interference studies showed that the genosensor has satisfactory selectivity since the hybridization with a non-specific probe resulted in a signal decrease (46% lower) when compared to the specific target.

Clinical isolates of the modern Mycobacterium tuberculosis lineage 4 evade host defense in human macrophages through eluding IL-1ß-induced autophagy.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily "ancient", and three "modern". The MTBC strains belonging to "modern" lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1ß, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1ß-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages.

Fluorescent Mycobacterium tuberculosis reporters: illuminating host-pathogen interactions.

The pathogenesis of Mycobacterium tuberculosis (Mtb) is intrinsically linked to its intimate and enduring interaction with its host, and understanding Mtb-host interactions at a molecular level is critical to attempts to decrease the significant burden of tuberculosis disease. The marked heterogeneity that exists in lesion progression and outcome during Mtb infection necessitates the development of methods that enable in situ analyses of Mtb biology and host response within the spatial context of tissue structure. Fluorescent reporter Mtb strains have thus come to the forefront as an approach with broad utility for the study of the Mtb-host interface, enabling visualization of the bacteria during infection, and contributing to the discovery of several facets such as non-uniformity in microenvironments and Mtb physiology in vivo, and their relation to the host immune response or therapeutic intervention. We review here the different types of fluorescent reporters and ways in which they have been utilized in Mtb studies, and expand on how they may further be exploited in combination with novel imaging and other methodologies to illuminate key aspects of Mtb-host interactions.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.