RESUMO
Sequence differences among the subtypes of Clostridioides difficile toxin TcdB (2,366 amino acids) are broadly distributed across the entire protein, with the notable exception of 76 residues at the protein's carboxy terminus. This sequence invariable region (SIR) is identical at the DNA and protein level among the TcdB variants, suggesting this string of amino acids has undergone selective pressure to prevent alterations. The functional role of the SIR domain in TcdB has not been determined. Analysis of a recombinantly constructed TcdB mutant lacking the SIR domain did not identify changes in TcdB's enzymatic or cytopathic activities. To further assess the SIR region, we constructed a C. difficile strain with the final 228 bp deleted from the tcdB gene, resulting in the production of a truncated form of TcdB lacking the SIR (TcdB2∆2291-2366). Using a combination of approaches, we found in the absence of the SIR sequence TcdB2∆2291-2366 retained cytotoxic activity but was not secreted from C. difficile. TcdB2∆2291-2366 was not released from the cell under autolytic conditions, indicating the SIR is involved in a more discrete step in toxin escape from the bacterium. Fractionation experiments combined with antibody detection found that TcdB2∆2291-2366 accumulates at the cell membrane but is unable to complete steps in secretion beyond this point. These data suggest conservation of the SIR domain across variants of TcdB could be influenced by the sequence's role in efficient escape of the toxin from C. difficile. IMPORTANCE: Clostridioides difficile is a leading cause of antibiotic associated disease in the United States. The primary virulence factors produced by C. difficile are two large glucosylating toxins TcdA and TcdB. To date, several sequence variants of TcdB have been identified that differ in various functional properties. Here, we identified a highly conserved region among TcdB subtypes that is required for release of the toxin from C. difficile. This study reveals a putative role for the longest stretch of invariable sequence among TcdB subtypes and provides new details regarding toxin release into the extracellular environment. Improving our understanding of the functional roles of the conserved regions of TcdB variants aids in the development of new, broadly applicable strategies to treat CDI.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Humanos , Regulação Bacteriana da Expressão Gênica , Sequência de Aminoácidos , AnimaisRESUMO
Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Centro Germinativo , Receptores CXCR4 , Animais , Receptores CXCR4/metabolismo , Receptores CXCR4/imunologia , Centro Germinativo/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Clostridioides difficile/imunologia , Clostridioides difficile/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos B/imunologia , Linfócitos B/metabolismo , Quimiocina CXCL12/metabolismo , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Memória Imunológica , Feminino , Formação de Anticorpos/imunologiaRESUMO
The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Humanos , Animais , Camundongos , Clostridioides difficile/genética , Via de Sinalização Hippo , Toxinas Bacterianas/metabolismo , Células HeLa , Clostridioides , RNA Mensageiro/metabolismo , Proteínas de Membrana/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismoRESUMO
Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.
Assuntos
Clostridioides difficile , Proteínas de Bactérias/metabolismo , Clostridioides , Clostridioides difficile/genética , Regulação Bacteriana da Expressão Gênica , Esporos BacterianosRESUMO
Group 3 innate lymphocytes (ILC3s) are rare immune cells localized in mucosal tissues, especially the gastrointestinal (GI) tract. Despite their rarity, they are a major source of the cytokine interleukin-22 (IL-22), which protects the GI epithelium during inflammation and infection. Although ILC3s have been demonstrated to be important for defense against Clostridioides difficile infection, the exact mechanisms through which they sense productive infection and become activated to produce IL-22 remain poorly understood. In this study, we identified a novel mechanism of ILC3 activation after exposure to C. difficile. Toxin B (TcdB) from C. difficile directly induced production of IL-22 in ILC3s, and this induction was dependent on the glucosyltransferase activity of the toxin, which inhibits small GTPases. Pharmacological inhibition of the small GTPase Cdc42 also enhanced IL-22 production in ILC3s, indicating that Cdc42 is a negative regulator of ILC3 activation. Further gene expression analysis revealed that treatment with TcdB modulated the expression of several inflammation-related genes in ILC3s. These findings demonstrate that C. difficile toxin-mediated inhibition of Cdc42 leads to the activation of ILC3s, providing evidence for how these cells are recruited into the immune response against the pathobiont.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Humanos , Imunidade Inata , Inflamação/metabolismo , LinfócitosRESUMO
The Gram-positive bacterium Clostridioides difficile is a primary cause of hospital-acquired diarrhea, threatening both immunocompromised and healthy individuals. An important aspect of defining mechanisms that drive C. difficile persistence and virulence relies on developing a more complete understanding of sporulation. C. difficile sporulation is the single determinant of transmission and complicates treatment and prevention due to the chemical and physical resilience of spores. By extension, the identification of druggable targets that significantly attenuate sporulation would have a significant impact on thwarting C. difficile infection. By use of a new CRISPR-Cas9 nickase genome editing methodology, stop codons were inserted early in the coding sequence for clpP1 and clpP2 to generate C. difficile mutants that no longer produced the corresponding isoforms of caseinolytic protease P (ClpP). The data show that genetic ablation of ClpP isoforms leads to altered sporulation phenotypes with the clpP1/clpP2 double mutant exhibiting asporogenic behavior. A small screen of known ClpP inhibitors in a fluorescence-based biochemical assay identified bortezomib as an inhibitor of C. difficile ClpP that produces dose-dependent inhibition of purified ClpP. Incubation of C. difficile cultures in the presence of bortezomib reveals antisporulation effects approaching that observed in the clpP1/clpP2 double mutant. This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small-molecule ClpP inhibitors as C. difficile antisporulating agents. IMPORTANCE Due to diverse roles of ClpP and the reliance of pathogens upon this system for infection, it has emerged as a target for antimicrobial development. Biology regulated by ClpP is organism dependent and has not been defined in Clostridioides difficile. This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small-molecule ClpP inhibitors as antisporulating agents. The identification of new approaches and/or drug targets that reduce C. difficile sporulation would be transformative and are expected to find high utility in prophylaxis, transmission attenuation, and relapse prevention. Discovery of the ClpP system as a major driver to sporulation also provides a new avenue of inquiry for advancing the understanding of sporulation.
Assuntos
Proteínas de Bactérias/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Bortezomib/farmacologia , Clostridioides difficile/química , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/microbiologia , Edição de Genes/métodos , Humanos , Mutação , Fenótipo , Isoformas de Proteínas/genética , Esporos Bacterianos/metabolismo , VirulênciaRESUMO
All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and, when conjugated to a protein carrier, induces a protective antibody response in animal models. Given that CD1d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18-/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.
Assuntos
Antígenos de Bactérias/imunologia , Clostridioides difficile/imunologia , Galactosilceramidas/imunologia , Imunoglobulina G/sangue , Células T Matadoras Naturais/imunologia , Polissacarídeos Bacterianos/imunologia , Animais , Anticorpos Antibacterianos/sangue , Feminino , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts.
Assuntos
Adjuvantes de Vacinas , Compostos de Alúmen , Vacinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Infecções por Clostridium/imunologia , Infecções por Clostridium/prevenção & controle , Células T Matadoras Naturais/imunologia , Adjuvantes de Vacinas/administração & dosagem , Adjuvantes de Vacinas/química , Animais , Vacinas Bacterianas/administração & dosagem , Biomarcadores , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Feminino , Imunização , Imunofenotipagem , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Células T Matadoras Naturais/metabolismoRESUMO
The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection. Here, we used a murine model of C. difficile disease recurrence to demonstrate that an initial infection induced a serum IgM and mucosal IgA response against the toxin, but a low serum IgG response, which is associated with a lack of protection against disease during reinfection. Infection induced a partial expansion of the T follicular helper cell compartment, essential for B cell memory responses, and, consistent with that, failed to significantly expand the memory B cell compartment. Further, infection failed to stimulate the memory B cell compartment in preimmunized mice, although they were protected against associated disease. These results delineate the key humoral immune events that follow primary and recurrent C. difficile infection and provide a compelling inverse correlation between B cell memory and disease recurrence.
Assuntos
Linfócitos B/imunologia , Clostridioides difficile/imunologia , Infecções por Clostridium/imunologia , Imunização , Imunoglobulina G/sangue , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Toxinas Bacterianas/imunologia , Infecções por Clostridium/microbiologia , Imunoglobulina A/metabolismo , Camundongos , Mucosa/metabolismoRESUMO
The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action.IMPORTANCEC. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr's mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Clostridioides difficile/fisiologia , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Sistemas CRISPR-Cas , Movimento , Mutação , Fenótipo , Filogenia , Transdução de Sinais , Esporos Bacterianos/genéticaRESUMO
Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769-1787, exhibited no detectable cytotoxicity. TcdB2Δ1769-1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769-1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769-1787 is a promising candidate for vaccination.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Proteínas Repressoras/imunologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Células CHO , Cricetulus , Glicosilação , Células HeLa , Humanos , Camundongos , Conformação Proteica , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Deleção de Sequência , VacinaçãoRESUMO
Caseinolytic protease P (ClpP) has emerged as a promising new target for antibacterial development. While ClpPs from single isoform expressing bacteria have been studied in detail, the function and regulation of systems with more than one ClpP homologue are still poorly understood. Herein, we present fundamental studies toward understanding the ClpP system in C. difficile, an anaerobic spore-forming pathogen that contains two chromosomally distant isoforms of ClpP. Examination of proteomic and genomic data suggest that ClpP1 is the primary isoform responsible for normal growth and virulence, but little is known about the function of ClpP2 or the context required for the formation of functional proteases. For the first time in a pathogenic bacterium, we demonstrate that both isoforms are capable of forming operative proteases. Interestingly, ClpP1 is the only homologue that possesses characteristic response to small molecule acyldepsipeptide activation. On the contrary, both ClpP1 and ClpP2 respond to cochaperone activation to degrade an ssrA-tagged substrate. These observations indicate that ClpP2 is less susceptible to acyldepsipeptide activation but retains the ability to interact with a known cochaperone. Homology models reveal no obvious characteristics that would allow one to predict less efficient acyldepsipeptide binding. The reported findings establish the uniqueness of the ClpP system in C. difficile, open new avenues of inquiry, and highlight the importance of more detailed structural, genetic, and biological characterization of the ClpP system in C. difficile.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Depsipeptídeos/química , Depsipeptídeos/farmacocinética , Endopeptidase Clp/química , Endopeptidase Clp/genética , Clostridioides difficile/enzimologia , Regulação Bacteriana da Expressão Gênica , Isoenzimas/genética , Proteólise , Proteômica , Homologia de Sequência de Aminoácidos , VirulênciaRESUMO
We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.