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Mycobacterium tuberculosis(Mtb) is a bacterial pathogen that causes tuberculosis, an infectious disease that inflicts major health and economic costs around the world1. Mtb encounters a diversity of environments during its lifecycle, and responds to these changing environments by reprogramming its transcriptional output2. However, the transcriptomic features of Mtb remain poorly characterized. In this work, we comprehensively profile the Mtb transcriptome using the SEnd-seq method that simultaneously captures the 5’ and 3’ ends of RNA3. Surprisingly, we find that the RNA coverage for most of the Mtb transcription units display a gradual drop-off within a 200-500 nucleotide window downstream of the transcription start site, yielding a massive number of incomplete transcripts with heterogeneous 3’ ends. We further show that the accumulation of these short RNAs is mainly due to the intrinsically low processivity of the Mtb transcription machinery rather than trans-acting factors such as Rho. Finally, we demonstrate that transcription-translation coupling plays a critical role in generating full-length protein-coding transcripts in Mtb. In sum, our results depict a mycobacterial transcriptome that is dominated by incomplete RNA products, suggesting a distinctive set of transcriptional regulatory mechanisms that could be exploited for new therapeutics.

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis . The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae ( Sven ), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σ A -holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX . These structures reveal that WhiB binds to domain 4 of σ A (σ A 4 ) of the σ A -holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the −35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σ A 4 housekeeping σ-factors and the −35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein–DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σ A -holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g.,Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g.,Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogenMycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show thatMtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand–DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausingE. coliNGN contacts the RNAP gate loop to inhibit swiveling and pausing ofMtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, directed predominantly to epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation. One Sentence Summary Hyper-focused antibody responses to variable antigenic coats allow pathogens to escape immune recognition.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (Rif(R)) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of Rif(R) RNAPs, including the most prevalent clinical Rif(R) RNAP substitution found in Mtb infected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the Rif(R) S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and ‘switching’ to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-A-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact. Trypanosoma brucei parasites switch their variant surface glycoprotein (VSG) coats to escape immune recognition. The crystal structure of VSG3 reveals that additional modifications, such as O-glycosylation, further expand antigenic variation and potentiate immune evasion.

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β′ subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli., Understanding of the mycobacterial transcription system is useful to the development of therapeutics against tuberculosis infection. Here the authors present the crystal structure of a complete M. smegmatis RNA polymerase open promoter complex that reveals unique features of the mycobacterial polymerase.

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket., eLife digest Tuberculosis (TB) is an infectious disease that affects over ten million people every year. The Mycobacterium tuberculosis bacteria that cause the disease spread through the air from one person to another and mainly infect the lungs. Although curable, TB is difficult to eradicate because it is remarkably widespread, with one third of the world’s population estimated to carry the bacteria. Treatment for TB involves a mix of antibiotics that should be taken for several months to a year. The number of multidrug-resistant TB cases, where the infection is not treatable by the common cocktail of antibiotics, is rapidly increasing. There is therefore a need to discover new drugs that can kill the M. tuberculosis bacteria. An antibiotic called fidaxomicin is used to treat intestinal infections. Although it can kill Mycobacterium tuberculosis cells in culture, it is not absorbed from the intestines to the blood and thus cannot reach the lungs to kill the bacteria. It may be possible to change the structure of the drug so that it can enter the bloodstream. Before this can be done, researchers need to understand exactly how fidaxomicin kills the bacteria so that they know which parts of the drug they can alter without making it less effective. Fidaxomicin kills bacterial cells by binding to an enzyme called RNA polymerase. The antibiotic prevents the enzyme from reading and ‘transcribing’ DNA to form molecules that are essential for life. To learn more about how fidaxomicin has this effect, Boyaci, Chen et al. used cryo-electron microscopy to look at structures of the M. tuberculosis RNA polymerase in different states, including when it was bound to fidaxomicin. The structures reveal the chemical details of the interactions between the RNA polymerase and the antibiotic. The two molecules bind to each other through a region of the RNA polymerase that is unique to M. tuberculosis and closely related bacteria. Fidaxomicin acts like a doorstop to jam the RNA polymerase in an open state that cannot bind to DNA and transcribe genes. Medicinal chemists could now build on these findings to develop new drugs that might treat TB, either by modifying fidaxomicin or designing new antibiotics that bind to the same region of the RNA polymerase. Because the fidaxomicin-binding region of the RNA polymerase is specific to M. tuberculosis new antibiotics could be tailored towards the bacteria that have a minimal effect on a patient’s normal gut bacteria.

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective againstMycobacterium tuberculosisRNAPin vitro, but clinical use of Fdx is limited to treatingClostridium difficileintestinal infections due to poor absorption. To enable structure-guided optimization of Fdx to treat tuberculosis, we report the 3.4 Å cryo-electron microscopy structure of a completeM. tuberculosisRNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin and explains its strong effect onM. tuberculosis. We present additional structures that define conformational states ofM. tuberculosisRNAP between the free apo-holenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on Fdx.

Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design., In bacteria, type III secretion systems (T3SS) allow the direct transport of protein across membranes, and related elements of a sorting platform facilitate hierarchical secretion of protein substrates. Here, the authors illustrate the mechanism of selective assembly of the T3SS sorting platforms in injectisome and flagellar systems.

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor inEscherichia colicells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. ThehslJgene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium.cysBmutants showed upregulation of thehslJ : : lacZgene fusion and exhibited increased novobiocin resistance. In this study thehslJtranscription start point and the corresponding putativeσ70promoter were determined. ThehslJpromoter region was defined by employing differenthslJ–lacZoperon fusions, and transcription of thehslJgene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor ofhslJtranscription and binds thehslJpromoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligandN-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression ofhslJtranscription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.

Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

Like many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen. By combining x-ray crystallography of SipA with electron microscopy and image analysis of SipA-actin filaments, we show that SipA functions as a “molecular staple,” in which a globular domain and two nonglobular “arms” mechanically stabilize the filament by tethering actin subunits in opposing strands. Deletion analysis of the tethering arms provides strong support for this model.

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.