Design and synthesis of Thieno[3, 2-b]pyridinone derivatives exhibiting potent activities against Mycobacterium tuberculosis in vivo by targeting Enoyl-ACP reductase.

In this study, a series of novel thieno [3, 2-b]pyridinone derivatives were designed and synthesized using a scaffold hopping strategy. Six compounds showed potent anti-mycobacterial activity (minimum inhibitory concentration (MIC) ≤ 1 µg/mL) against Mycobacterium tuberculosis (Mtb) UAlRa. Compound 6c displayed good activity against Mtb UAlRv (MIC = 0.5-1 µg/mL). Compounds 6c and 6i also showed activity against Mtb UAlRa in macrophages and exhibited low cytotoxicity against LO-2 cells. The selected compounds displayed a narrow antibacterial spectrum, with no activity against representative Gram-positive, Gram-negative bacteria, as well as fungi. Furthermore, compound 6c demonstrated favorable oral pharmacokinetic properties with a T1/2 value of 47.99 h and exhibited good in vivo activity in an acute mouse model of tuberculosis (TB). The target of compound 6c was identified as a NADH-dependent enoyl-acyl carrier protein reductase (InhA) by genome sequencing of spontaneously compound 6c-resistant Mtb mutants, indicating that compound 6c may not require activation and can directly target InhA. In vitro antimicrobial assays against a recombinant M. smegmatis overexpressing the Mtb-InhA, along with InhA inhibition assays, confirmed that InhA is the target of thieno [3, 2-b]pyridinone derivatives. Overall, this study identified thieno [3, 2-b]pyridinone scaffold as a novel chemotype that is promising for the development of anti-TB agents.

Engineered probiotic Escherichia coli elicits immediate and long-term protection against influenza A virus in mice.

Influenza virus infection remains a major global health problem and requires a universal vaccine with broad protection against different subtypes as well as a rapid-response vaccine to provide immediate protection in the event of an epidemic outbreak. Here, we show that intranasal administration of probiotic Escherichia coli Nissle 1917 activates innate immunity in the respiratory tract and provides immediate protection against influenza virus infection within 1 day. Based on this vehicle, a recombinant strain is engineered to express and secret five tandem repeats of the extracellular domain of matrix protein 2 from different influenza virus subtypes. Intranasal vaccination with this strain induces durable humoral and mucosal responses in the respiratory tract, and provides broad protection against the lethal challenge of divergent influenza viruses in female BALB/c mice. Our findings highlight a promising delivery platform for developing mucosal vaccines that provide immediate and sustained protection against respiratory pathogens.

A broadly protective antibody targeting glycoprotein Gn inhibits severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes severe viral hemorrhagic fever and thrombocytopenia syndrome with a fatality rate of up to 30%. No licensed vaccines or therapeutics are currently available for humans. Here, we develop seven monoclonal antibodies (mAbs) against SFTSV surface glycoprotein Gn. Mechanistic studies show that three neutralizing mAbs (S2A5, S1G3, and S1H7) block multiple steps during SFTSV infection, including viral attachment and membrane fusion, whereas another neutralizing mAb (B1G11) primarily inhibits the viral attachment step. Epitope binning and X-ray crystallographic analyses reveal four distinct antigenic sites on Gn, three of which have not previously been reported, corresponding to domain I, domain II, and spanning domain I and domain II. One of the most potent neutralizing mAbs, S2A5, binds to a conserved epitope on Gn domain I and broadly neutralizes infection of six SFTSV strains corresponding to genotypes A to F. A single dose treatment of S2A5 affords both pre- and post-exposure protection of mice against lethal SFTSV challenge without apparent weight loss. Our results support the importance of glycoprotein Gn for eliciting a robust humoral response and pave a path for developing prophylactic and therapeutic antibodies against SFTSV infection.

The Copper Efflux Regulator (CueR).

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis.

Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections. IMPORTANCE: Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

Cryo-EM Structure of Porphyromonas gingivalis RNA Polymerase.

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in ß and ß' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development.

Structural basis for transcription activation by the nitrate-responsive regulator NarL.

Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.

Structure and molecular mechanism of bacterial transcription activation.

Transcription activation is an important checkpoint of regulation of gene expression which occurs in response to different intracellular and extracellular signals. The key elements in this signal transduction process are transcription activators, which determine when and how gene expression is activated. Recent structural studies on a considerable number of new transcription activation complexes (TACs) revealed the remarkable mechanistic diversity of transcription activation mediated by different factors, necessitating a review and re-evaluation of the transcription activation mechanisms. In this review, we present a comprehensive summary of transcription activation mechanisms and propose a new, elaborate, and systematic classification of transcription activation mechanisms, primarily based on the structural features of diverse TAC components.

Association between mutations in a thyX-hsdS.1 region and para-aminosalicylic acid resistance in Mycobacterium tuberculosis clinical isolates.

Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis agent for decades, its mechanisms of resistance are still not thoroughly understood. Previously, sporadic studies showed that certain mutations in the thyX-hsdS.1 region caused PAS resistance in M. tuberculosis, but a comprehensive analysis is lacking. Recently, we found a G-10A mutation in thyX-hsdS.1 in a PAS-resistant clinical isolate, but it did not cause PAS resistance. SNPs in thyX-hsdS.1 in 6550 clinical isolates were analyzed, and 153 SNPs were identified. C-16 T was the most common SNP identified (54.25%, 83/153), followed by C-4T (7.19%, 11/153) and G-9A (6.54%, 10/153). Subsequently, the effects of those SNPs on the promoter activity of thyX were tested, and the results showed that mutations C-1T, G-3A, C-4T, C-4G, G-7A, G-9A, C-16T, G-18C, and C-19G led to increased promoter activity compared with the wild-type sequence, but other mutations did not. Then, thyX and wild-type thyX-hsdS.1, or thyX-hsdS.1 containing specific SNPs, were overexpressed in M. tuberculosis H37Ra. The results showed that mutations resulting in increased promoter activity also caused PAS resistance. Moreover, the results of an electrophoretic mobility shift assay showed that thyX-hsdS.1 containing the C-16T mutation had a higher binding capacity to RNA polymerase than did the wild-type sequence. Taken together, our data demonstrated that among the SNPs identified in thyX-hsdS.1 of M. tuberculosis clinical isolates, only those able to increase the promoter activity of thyX caused PAS resistance and therefore can be considered as molecular markers for PAS resistance.

Rational design of a genome-based insulated system in Escherichia coli facilitates heterologous uricase expression for hyperuricemia treatment.

Hyperuricemia is a prevalent disease worldwide that is characterized by elevated urate levels in the blood owing to purine metabolic disorders, which can result in gout and comorbidities. To facilitate the treatment of hyperuricemia through the uricolysis, we engineered a probiotic Escherichia coli Nissle 1917 (EcN) named EcN C6 by inserting an FtsP-uricase cassette into an "insulated site" located between the uspG and ahpF genes. Expression of FtsP-uricase in this insulated region did not influence the probiotic properties or global gene transcription of EcN but strongly increased the enzymatic activity for urate degeneration, suggesting that the genome-based insulated system is an ideal strategy for EcN modification. Oral administration of EcN C6 successfully alleviated hyperuricemia, related symptoms and gut microbiota in a purine-rich food-induced hyperuricemia rat model and a uox-knockout mouse model. Together, our study provides an insulated site for heterologous gene expression in EcN strain and a recombinant EcN C6 strain as a safe and effective therapeutic candidate for hyperuricemia treatment.

Roles of zinc-binding domain of bacterial RNA polymerase in transcription.

Transcription is an essential and multistep process carried out by RNA polymerase (RNAP). In bacterial RNAP, in addition to the catalytic core domain, multiple other conserved domains are also identified to play regulatory roles in transcription. One such domain is the zinc-binding domain (ZBD) located at the N terminus of the largest subunit ß' in bacterial RNAP, whose homolog is also reported in eukaryotic RNAPs. Recent structural and biochemical studies have revealed various key roles of the conserved ß' ZBD during different steps of transcription. In this review, we summarize recent progress on the regulatory roles of this ß' ZBD in bacterial transcription.

A Feedback Regulatory Loop Containing McdR and WhiB2 Controls Cell Division and DNA Repair in Mycobacteria.

Cell division must be coordinated with DNA repair, which is strictly regulated in response to different drugs and environmental stresses in bacteria. However, the mechanisms by which mycobacteria orchestrate these two processes remain largely uncharacterized. Here, we report a regulatory loop between two essential mycobacterial regulators, McdR (Rv1830) and WhiB2, in coordinating the processes of cell division and DNA repair. McdR inhibits cell division-associated whiB2 expression by binding to the AATnACAnnnnTGTnATT motif in the promoter region. Furthermore, McdR overexpression simultaneously activates imuAB and dnaE2 expression to promote error-prone DNA repair, which facilitates genetic adaptation to stress conditions. Through a feedback mechanism, WhiB2 activates mcdR expression by binding to the cGACACGc motif in the promoter region. Importantly, analyses of mutations in clinical Mycobacterium tuberculosis strains indicate that disruption of this McdR-WhiB2 feedback regulatory loop influences expression of both cell growth- and DNA repair-associated genes, which further supports the contribution of McdR-WhiB2 regulatory loop in regulating mycobacterial cell growth and drug resistance. This highly conserved feedback regulatory loop provides fresh insight into the link between mycobacterial cell growth control and stress responses. IMPORTANCE Drug-resistant M. tuberculosis poses a threat to the control and prevention of tuberculosis (TB) worldwide. Thus, there is a need to identify the mechanisms enabling M. tuberculosis to adapt and grow under drug-induced stress. Rv1830 has been shown to be associated with drug resistance in M. tuberculosis, but its mechanisms have not yet been elucidated. Here, we reveal a regulatory role of Rv1830, which coordinates cell division and DNA repair in mycobacteria, and rename it McdR (mycobacterial cell division regulator). An increase in McdR levels represses the expression of cell division-associated whiB2 but activates the DNA repair-associated, error-prone enzymes ImuA/B and DnaE2, which in turn facilitates adaptation to stress responses and drug resistance. Furthermore, WhiB2 activates the transcription of mcdR to form a conserved regulatory loop. These data provide new insights into the mechanisms controlling mycobacterial cell growth and stress responses.

Molecular Epidemiology and Risk Factors of Carbapenem-Resistant Klebsiella Pneumoniae Bloodstream Infections in Wuhan, China.

OBJECTIVE: The clinical characteristics and microbiological data of patients with K. pneumoniae bloodstream infections (BSI) from January 2018 to December 2020 were retrospectively analyzed to study the molecular epidemiology of Carbapenem-resistant Klebsiella pneumoniae (CRKP). We also aimed to identify the risk factors for the development of CRKP BSI. METHODS: This retrospective study was conducted at Renmin Hospital of Wuhan University from January 2018 to December 2020. The date of non-duplicate K. pneumoniae isolates isolated from blood samples was identified using the microbiology laboratory database. The data from patients diagnosed with K. pneumoniae BSI were collected and analyzed. RESULTS: From 2018 to 2020, there were 510 non-duplicated K. pneumoniae blood isolates, mainly distributed in the intensive care unit (ICU) (28.4%), that were identified in our research. These cases included 77 strains of CRKP and 433 strains of carbapenem-susceptible K. pneumoniae (CSKP). The resistance rate of K. pneumoniae to meropenem and imipenem increased from 11.2% in 2018 to 27.1% in 2020. Moreover, Compared with CSKP, all CRKP isolates showed multi-resistance to tested antibiotics. The phylogenetic analysis showed that the CRKP isolates could be grouped into four major clades, and multilocus sequence typing revealed that the isolates had considerable clonality. Overall, 8 sequence types (STs) of CRKP were detected, of which ST11 comprised the majority and clustered into clade 3. The most prevalent carbapenemase gene was blaKPC (87%) among the CRKP isolates, followed by blaNDM (9.1%) and blaIMP (1.3%). A total of 74 (16.6%) patients with CRKP BSI and 373 (83.4%) patients with CSKP BSI were categorized as the case and control groups. The mortality in the CRKP group was 44.6%, and 11.5% in CSKP group (P<0.001). A multivariate analysis that a long hospital stay before BSI (OR=1.42, 95% CI 1.02-4.31, P=0.011), ICU hospitalization history (OR=3.30, 95% CI 1.35-8.05, P=0.002), and prior use of carbapenems (OR=3.33, 95% CI 1.29-7.27, P=0.001) and antifungals (OR=2.81, 95% CI 1.24-6.04, P<0.001) were independent risk factors for CRKP BSI. CONCLUSION: ST11 is the predominant type of CRKP mediating inter-hospital transmission, and blaKPC is the main carbapenemase gene harboured by CRKP blood isolates. ICU stay, prolonged hospitalization before BSI, and prior use of carbapenems and antifungals were independent risk factors for acquiring CRKP BSI. Our study may provide insights into early infection control practices.

Structural basis for activation of Swi2/Snf2 ATPase RapA by RNA polymerase.

RapA is a bacterial RNA polymerase (RNAP)-associated Swi2/Snf2 ATPase that stimulates RNAP recycling. The ATPase activity of RapA is autoinhibited by its N-terminal domain (NTD) but activated with RNAP bound. Here, we report a 3.4-Å cryo-EM structure of Escherichia coli RapA-RNAP elongation complex, in which the ATPase active site of RapA is structurally remodeled. In this process, the NTD of RapA is wedged open by RNAP ß' zinc-binding domain (ZBD). In addition, RNAP ß flap tip helix (FTH) forms extensive hydrophobic interactions with RapA ATPase core domains. Functional assay demonstrates that removing the ZBD or FTH of RNAP significantly impairs its ability to activate the ATPase activity of RapA. Our results provide the structural basis of RapA ATPase activation by RNAP, through the active site remodeling driven by the ZBD-buttressed large-scale opening of NTD and the direct interactions between FTH and ATPase core domains.

Structural basis of copper-efflux-regulator-dependent transcription activation.

The copper efflux regulator (CueR), a representative member of mercury resistance regulator (MerR) family metalloregulators, controls expression of copper homeostasis-regulating genes in bacteria. The mechanism of transcription activation by CueR and other MerR family regulators is bending the spacer domain of promoter DNA. Here, we report the cryo-EM structures of the intact CueR-dependent transcription activation complexes. The structures show that CueR dimer bends the 19-bp promoter spacer to realign the -35 and -10 elements for recognition by σ70-RNA polymerase holoenzyme and reveal a previously unreported interaction between the DNA-binding domain (DBD) from one CueR subunit and the σ70 nonconserved region (σNCR). Functional studies have shown that the CueR-σNCR interaction plays an auxiliary role in CueR-dependent transcription, assisting the activation mechanism of bending promoter DNA by CueR dimer. Because DBDs are highly conserved in sequence and structure, this transcription-activating mechanism could be generally used by MerR family regulators.

LcrQ Coordinates with the YopD-LcrH Complex To Repress lcrF Expression and Control Type III Secretion by Yersinia pseudotuberculosis.

Human-pathogenic Yersinia species employ a plasmid-encoded type III secretion system (T3SS) to negate immune cell function during infection. A critical element in this process is the coordinated regulation of T3SS gene expression, which involves both transcriptional and posttranscriptional mechanisms. LcrQ is one of the earliest identified negative regulators of Yersinia T3SS, but its regulatory mechanism is still unclear. In a previous study, we showed that LcrQ antagonizes the activation role played by the master transcriptional regulator LcrF. In this study, we confirm that LcrQ directly interacts with LcrH, the chaperone of YopD, to facilitate the negative regulatory role of the YopD-LcrH complex in repressing lcrF expression at the posttranscriptional level. Negative regulation is strictly dependent on the YopD-LcrH complex, more so than on LcrQ. The YopD-LcrH complex helps to retain cytoplasmic levels of LcrQ to facilitate the negative regulatory effect. Interestingly, RNase E and its associated protein RhlB participate in this negative regulatory loop through a direct interaction with LcrH and LcrQ. Hence, we present a negative regulatory loop that physically connects LcrQ to the posttranscriptional regulation of LcrF, and this mechanism incorporates RNase E involved in mRNA decay. IMPORTANCE All three human-pathogenic Yesinia species, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis, employ a plasmid-encoded T3SS to target immunomodulatory effectors into host immune cells. Several plasmid-encoded regulators influence T3SS control, including the master transcriptional activator LcrF, the posttranscriptional repressor YopD, and the unassigned negative regulatory factor LcrQ. Since LcrQ lacks any obvious DNA or RNA binding domains, its regulatory mechanism might be special. In this study, we screened for proteins that directly engaged with LcrQ. We found that LcrQ cooperates with LcrH of the YopD-LcrH complex to aid in the posttranscriptional repression of lcrF expression. This negative-control loop also involved the mRNA decay factor RNase E and its associated RhlB protein, which were recruited to the regulatory complex by both LcrQ and LcrH. Hence, we identify interacting components of LcrQ that shed new light on a mechanism inhibiting T3SS production and biogenesis.

Label-Free Comparative Proteomics of Differentially Expressed Mycobacterium tuberculosis Protein in Rifampicin-Related Drug-Resistant Strains.

Rifampicin (RIF) is one of the most important first-line anti-tuberculosis (TB) drugs, and more than 90% of RIF-resistant (RR) Mycobacterium tuberculosis clinical isolates belong to multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. In order to identify specific candidate target proteins as diagnostic markers or drug targets, differential protein expression between drug-sensitive (DS) and drug-resistant (DR) strains remains to be investigated. In the present study, a label-free, quantitative proteomics technique was performed to compare the proteome of DS, RR, MDR, and XDR clinical strains. We found iniC, Rv2141c, folB, and Rv2561 were up-regulated in both RR and MDR strains, while fadE9, espB, espL, esxK, and Rv3175 were down-regulated in the three DR strains when compared to the DS strain. In addition, lprF, mce2R, mce2B, and Rv2627c were specifically expressed in the three DR strains, and 41 proteins were not detected in the DS strain. Functional category showed that these differentially expressed proteins were mainly involved in the cell wall and cell processes. When compared to the RR strain, Rv2272, smtB, lpqB, icd1, and folK were up-regulated, while esxK, PPE19, Rv1534, rpmI, ureA, tpx, mpt64, frr, Rv3678c, esxB, esxA, and espL were down-regulated in both MDR and XDR strains. Additionally, nrp, PPE3, mntH, Rv1188, Rv1473, nadB, PPE36, and sseA were specifically expressed in both MDR and XDR strains, whereas 292 proteins were not identified when compared to the RR strain. When compared between MDR and XDR strains, 52 proteins were up-regulated, while 45 proteins were down-regulated in the XDR strain. 316 proteins were especially expressed in the XDR strain, while 92 proteins were especially detected in the MDR strain. Protein interaction networks further revealed the mechanism of their involvement in virulence and drug resistance. Therefore, these differentially expressed proteins are of great significance for exploring effective control strategies of DR-TB.

Structural visualization of transcription activated by a multidrug-sensing MerR family regulator.

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.

Genome-scale analyses of transcriptional start sites in Mycobacterium marinum under normoxic and hypoxic conditions.

BACKGROUND: Hypoxic stress plays a critical role in the persistence of Mycobacterium tuberculosis (Mtb) infection, but the mechanisms underlying this adaptive response remain ill defined. MATERIAL AND METHODS: In this study, using M. marinum as a surrogate, we analyzed hypoxic responses at the transcriptional level by Cappable-seq and regular RNA-seq analyses. RESULTS: A total of 6808 transcriptional start sites (TSSs) were identified under normoxic and hypoxic conditions. Among these TSSs, 1112 were upregulated and 1265 were downregulated in response to hypoxic stress. Using SigE-recognized consensus sequence, we identified 59 SigE-dependent promoters and all were upregulated under hypoxic stress, suggesting an important role for SigE in this process. We also compared the performance of Cappable-seq and regular RNA-seq using the same RNA samples collected from normoxic and hypoxic conditions, and confirmed that Cappable-seq is a valuable approach for global transcriptional regulation analyses. CONCLUSIONS: Our results provide insights and information for further characterization of responses to hypoxia in mycobacteria, and prove that Cappable-seq is a valuable approach for global transcriptional studies in mycobacteria.