RESUMEN
The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.
Asunto(s)
Proteínas Bacterianas , Borrelia burgdorferi , Regulación Bacteriana de la Expresión Génica , Estabilidad del ARN , Proteínas de Unión al ARN , Factor sigma , Factor sigma/metabolismo , Factor sigma/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Estabilidad del ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Regiones no Traducidas 5' , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Polimerasa Sigma 54/metabolismo , ARN Polimerasa Sigma 54/genéticaRESUMEN
PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/metabolismo , Grupo Borrelia Burgdorferi/genética , Enfermedad de Lyme/genética , ARN/metabolismoRESUMEN
We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD. Using a broad metabolomics approach, we found that GpsA serves as a dominant regulator of NADH and glycerol-3-phosphate levels in vitro, metabolic intermediates that reflect the cellular redox potential and serve as a precursor for lipid and lipoprotein biosynthesis, respectively. Additionally, GpsA was required for survival under nutrient stress, regulated overall reductase activity and controlled B. burgdorferi morphology in vitro. Furthermore, during in vitro nutrient stress, both glycerol and N-acetylglucosamine were bactericidal to B. burgdorferi in a GlpD-dependent manner. This study is also the first to identify a suppressor mutation in B. burgdorferi: a glpD deletion restored the wild-type phenotype to the pleiotropic gpsA mutant, including murine infectivity by needle inoculation at high doses, survival under nutrient stress, morphological changes and the metabolic imbalance of NADH and glycerol-3-phosphate. These results illustrate how basic metabolic functions that are dispensable for in vitro growth can be essential for in vivo infectivity of B. burgdorferi and may serve as attractive therapeutic targets.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Enfermedad de Lyme , Garrapatas , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/genética , Glicerolfosfato Deshidrogenasa/metabolismo , Ratones , NAD/metabolismo , Oxidación-Reducción , Fosfatos/metabolismoRESUMEN
Despite their ubiquitous nature, few antisense RNAs have been functionally characterized, and this class of RNAs is considered by some to be transcriptional noise. Here, we report that an antisense RNA (asRNA), aMEF (antisense mazEF), functions as a dual regulator for the type II toxin-antitoxin (TA) system mazEF. Unlike type I TA systems and many other regulatory asRNAs, aMEF stimulates the synthesis and translation of mazEF rather than inhibition and degradation. Our data indicate that a double-stranded RNA intermediate and RNase III are not necessary for aMEF-dependent regulation of mazEF expression. The lack of conservation of asRNA promoters has been used to support the hypothesis that asRNAs are spurious transcriptional noise and nonfunctional. We demonstrate that the aMEF promoter is active and functional in Escherichia coli despite poor sequence conservation, indicating that the lack of promoter sequence conservation should not be correlated with functionality. IMPORTANCE Next-generation RNA sequencing of numerous organisms has revealed that transcription is widespread across the genome, termed pervasive transcription, and does not adhere to annotated gene boundaries. The function of pervasive transcription is enigmatic and has generated considerable controversy as to whether it is transcriptional noise or biologically relevant. Antisense transcription is one class of pervasive transcription that occurs from the DNA strand opposite an annotated gene. Relatively few pervasively transcribed asRNAs have been functionally characterized, and their regulatory roles or lack thereof remains unknown. It is important to study examples of these asRNAs and determine if they are functional regulators. In this study, we elucidate the function of an asRNA (aMEF) demonstrating that pervasive transcripts can be functional.
Asunto(s)
ARN sin Sentido , Sistemas Toxina-Antitoxina , ARN sin Sentido/genética , Escherichia coli/genética , Regiones Promotoras Genéticas , Expresión Génica , Regulación Bacteriana de la Expresión GénicaRESUMEN
Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.
RESUMEN
Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.
Asunto(s)
Borrelia burgdorferi/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme/microbiología , Transcriptoma , Adaptación Fisiológica , Animales , Vectores Artrópodos/microbiología , Genes Bacterianos , Interacciones Huésped-Patógeno , Humanos , Enfermedad de Lyme/transmisión , Garrapatas/microbiologíaRESUMEN
Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi.
Asunto(s)
Borrelia burgdorferi/genética , ARN Pequeño no Traducido/metabolismo , Tropismo/genética , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidad , Regulación Bacteriana de la Expresión Génica/genética , Biblioteca de Genes , Genoma Bacteriano , Enfermedad de Lyme/microbiología , Proteómica , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Transcriptoma/genética , VirulenciaRESUMEN
RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. Here we show that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein (PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, we identify a conserved set of Shep/SUP-26 target RNAs that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Metamorfosis Biológica/genética , Morfogénesis/fisiología , Neurogénesis/genética , Proteínas de Unión al ARN/fisiología , Ribonucleoproteínas/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by RelBbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that RelBbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were RelBbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5' untranslated region sRNAs. RelBbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.
Asunto(s)
Borrelia burgdorferi/genética , Borrelia burgdorferi/fisiología , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/análisis , Transcriptoma , Estrés FisiológicoRESUMEN
The rRNA genes of Borrelia (Borreliella) burgdorferi are unusually organized; the spirochete has a single 16S rRNA gene that is more than 3 kb from a tandem pair of 23S-5S rRNA operons. We generated an rnc null mutant in B. burgdorferi that exhibits a pleiotropic phenotype, including decreased growth rate and increased cell length. Here, we demonstrate that endoribonuclease III (RNase III) is, as expected, involved in processing the 23S rRNA in B. burgdorferi The 5' and 3' ends of the three rRNAs were determined in the wild type and rncBb mutants; the results suggest that RNase III in B. burgdorferi is required for the full maturation of the 23S rRNA but not for the 5S rRNA nor, curiously, for the 16S rRNA.IMPORTANCE Lyme disease, the most common tick-borne zoonosis in the Northern Hemisphere, is caused by the bacterium Borrelia (Borreliella) burgdorferi, a member of the deeply branching spirochete phylum. B. burgdorferi carries a limited suite of ribonucleases, enzymes that cleave RNA during processing and degradation. Several ribonucleases, including RNase III, are involved in the production of ribosomes, which catalyze translation and are a major target of antibiotics. This is the first study to dissect the role of an RNase in any spirochete. We demonstrate that an RNase III mutant is viable but has altered processing of rRNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/enzimología , ARN Bacteriano/metabolismo , ARN Ribosómico 23S/metabolismo , Ribonucleasa III/metabolismo , Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Humanos , Enfermedad de Lyme/microbiología , Operón , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Ribonucleasa III/genéticaRESUMEN
In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.
Asunto(s)
Aptámeros de Nucleótidos/genética , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Técnica SELEX de Producción de Aptámeros , Terminación de la Transcripción Genética , Aptámeros de Nucleótidos/metabolismo , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Nicotinamida-Nucleótido Adenililtransferasa/genética , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Sistemas de Lectura Abierta , Ribosomas/metabolismo , Factores de TiempoRESUMEN
Borrelia (Borreliella) burgdorferi and closely related genospecies are the causative agents of Lyme disease, the most common tick-borne disease north of the equator. The bacterium, a member of the spirochete phylum, is acquired by a tick vector that feeds on an infected vertebrate host and is transmitted to another vertebrate during subsequent feeding by the next tick stage. The precise navigation of this enzootic cycle entails the regulation of genes required for these two host-specific phases as well as the transitions between them. Recently, an expansive swath of small RNAs has been identified in B. burgdorferi and likely many, if not most, are involved in regulating gene expression. Regardless, with only a few exceptions, the functions of these RNAs are completely unknown. However, several state-of-the-art approaches are available to identify the targets of these RNAs and provide insight into their role in the enzootic cycle and infection.
Asunto(s)
Borrelia burgdorferi/fisiología , ARN Pequeño no Traducido/fisiología , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Humanos , Enfermedad de Lyme/microbiología , ARN sin Sentido/genética , ARN sin Sentido/fisiología , ARN Pequeño no Traducido/genéticaRESUMEN
BACKGROUND: Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue-specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. RESULTS: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. CONCLUSIONS: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper-mediated RNA regulation. Developmental Dynamics 246:610-624, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Enfermedades Vasculares Periféricas/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hemodinámica/genética , Hemodinámica/fisiología , Metamorfosis Biológica/genética , Metamorfosis Biológica/fisiología , Enfermedades Vasculares Periféricas/genética , Proteínas de Unión al ARN/genética , Piel/citología , Piel/metabolismoRESUMEN
BACKGROUND: Transmission of Borrelia burgdorferi from its tick vector to a vertebrate host requires extensive reprogramming of gene expression. Small regulatory RNAs (sRNA) have emerged in the last decade as important regulators of bacterial gene expression. Despite the widespread observation of sRNA-mediated gene regulation, only one sRNA has been characterized in the Lyme disease spirochete B. burgdorferi. We employed an sRNA-specific deep-sequencing approach to identify the small RNA transcriptome of B. burgdorferi at both 23 °C and 37 °C, which mimics in vitro the transmission from the tick vector to the mammalian host. RESULTS: We identified over 1000 sRNAs in B. burgdorferi revealing large amounts of antisense and intragenic sRNAs, as well as characteristic intergenic and 5' UTR-associated sRNAs. A large fraction of the novel sRNAs (43%) are temperature-dependent and differentially expressed at the two temperatures, suggesting a role in gene regulation for adaptation during transmission. In addition, many genes important for maintenance of Borrelia during its enzootic cycle are associated with antisense RNAs or 5' UTR sRNAs. RNA-seq data were validated for twenty-two of the sRNAs via Northern blot analyses. CONCLUSIONS: Our study demonstrates that sRNAs are abundant and differentially expressed by environmental conditions suggesting that gene regulation via sRNAs is a common mechanism utilized in B. burgdorferi. In addition, the identification of antisense and intragenic sRNAs impacts the broadly used loss-of-function genetic approach used to study gene function and increases the coding potential of a small genome. To facilitate access to the analyzed RNA-seq data we have set-up a website at http://www.cibiv.at/~niko/bbdb/ that includes a UCSC browser track hub. By clicking on the respective link, researchers can interactively inspect the data in the UCSC genome browser (Kent et al., Genome Res 12:996-1006, 2002).
Asunto(s)
Borrelia burgdorferi/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , ARN Pequeño no Traducido/genética , Temperatura , Transcriptoma , Biología Computacional/métodos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedad de Lyme/microbiología , Sistemas de Lectura Abierta , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5' end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.
Asunto(s)
Borrelia burgdorferi/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme/microbiología , Transcriptoma , Animales , Expresión Génica , Genes Reporteros , Genoma Bacteriano , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Sitio de Iniciación de la Transcripción , Regiones no TraducidasRESUMEN
Two outer membrane protein (OMP) transport systems in diderm bacteria assist in assembly and export of OMPs. These two systems are the ß-barrel assembly machine (BAM) complex and the translocation and assembly module (TAM). The BAM complex consists of the OMP component BamA along with several outer membrane associated proteins. The TAM also consists of an OMP, designated TamA, and a single inner membrane (IM) protein, TamB. Together TamA and TamB aid in the secretion of virulence-associated OMPs. In this study we characterized the hypothetical protein BB0794 in Borrelia burgdorferi. BB0794 contains a conserved DUF490 domain, which is a motif found in all TamB proteins. All spirochetes lack a TamA ortholog, but computational and physicochemical characterization of BB0794 revealed it is a TamB ortholog. Interestingly, BB0794 was observed to interact with BamA and a BB0794 regulatable mutant displayed altered cellular morphology and antibiotic sensitivity. The observation that B. burgdorferi contains a TamB ortholog that interacts with BamA and is required for proper outer membrane biogenesis not only identifies a novel role for TamB-like proteins, but also may explain why most diderms harbor a TamB-like protein while only a select group encodes TamA.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Borrelia burgdorferi/metabolismo , Proteínas de Unión al ADN/metabolismo , Dominios Proteicos , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; relBbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.
Asunto(s)
Vectores Arácnidos/microbiología , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/fisiología , Regulación Bacteriana de la Expresión Génica , Ixodes/microbiología , Pirofosfatasas/metabolismo , Estrés Fisiológico , Animales , Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/crecimiento & desarrollo , Borrelia burgdorferi/ultraestructura , Tracto Gastrointestinal/microbiología , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Ixodes/fisiología , Larva/microbiología , Larva/fisiología , Viabilidad Microbiana , Microscopía Electrónica de Rastreo , Mutación , Ninfa/microbiología , Ninfa/fisiología , Operón , Pirofosfatasas/genética , Regulón , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , TranscriptomaRESUMEN
Pervasive, or genome-wide, transcription has been reported in all domains of life. In bacteria, most pervasive transcription occurs antisense to protein-coding transcripts, although recently a new class of pervasive RNAs was identified that originates from within annotated genes. Initially considered to be non-functional transcriptional noise, pervasive transcription is increasingly being recognized as important in regulating gene expression. The function of pervasive transcription is an extensively debated question in the field of transcriptomics and regulatory RNA biology. Here, we highlight the most recent contributions addressing the purpose of pervasive transcription in bacteria and discuss their implications.
Asunto(s)
ARN Bacteriano/metabolismo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , Procesamiento Postranscripcional del ARN , ARN sin Sentido/metabolismo , Ribonucleasa III/metabolismo , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
Hfq is a global regulator of gene expression in bacteria undergoing adaptation to changing environmental conditions. Its major function is to promote RNA-RNA interactions between regulatory small RNAs (sRNAs) and their target mRNAs. Previously, we demonstrated that Hfq binds many antisense RNAs (asRNAs) in vitro and hypothesized that Hfq may play a role in regulating gene expression via asRNAs. To investigate the E. coli Hfq-binding transcriptome in more detail, we co-immunoprecipitated and deep-sequenced RNAs bound to Hfq in vivo. We detected many new Hfq-binding sRNAs and observed that almost 300 mRNAs bind to Hfq. Among these, several are known to be sRNA targets. We identified 25 novel RNAs, which are transcribed from within protein coding regions and named them intragenic RNAs (intraRNAs). Furthermore, 67 asRNAs were co-immunoprecipitated with Hfq, demonstrating that Hfq binds antisense transcripts in vivo. Northern blot analyses confirmed the deep-sequencing results and demonstrated that many of the novel Hfq-binding RNAs identified are regulated by Hfq.