The transcriptional landscape of Chlamydia pneumoniae

Albrecht, Marco; Sharma, Cynthia M.; Dittrich, Marcus; Müller, Tobias; Reinhardt, Richard; Vogel, Jörg; Rudel, Thomas

doi:10.1186/gb-2011-12-10-r98

Cited by 73 publications

(55 citation statements)

References 58 publications

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“…Since many TS mutations are dominant, it also cannot be immediately differentiated if TS alleles are cis-acting or trans-acting. The TS alleles mapped in this study are not in known ncRNA regions (54)(55)(56), and missense mutations usually do not cause polar effects. Another limitation is that the phenotypes of the TS mutants need to be compared to those of the correspond- (A) Burst sizes were calculated by comparing the number of inclusions TS isolates formed in primary infections to the number formed in secondary infections performed with harvests from the primary infections.…”

Section: Discussionmentioning

confidence: 90%

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

et al. 2016

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Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperatureshift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCEStudy of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms. Chlamydiae are obligate intracellular pathogens that share a characteristic biphasic developmental cycle where these organisms alternate between extracellular, infectious elementary body (EB) and intracellular, replicative reticulate body (RB) forms (1). Multiple species of the family Chlamydiaceae, including Chlamydia trachomatis, C. pneumoniae, and C. psittaci, are common or incidental pathogens of humans (2). Despite their medical importance, a lack of tools for genetic manipulation of these organisms was a major impediment in chlamydial research until recently (3, 4).Only ϳ60% of Chlamydiaceae genes share significant ...

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Section: Discussionmentioning

confidence: 90%

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

et al. 2016

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“…The resulting lncRNA products might simply represent byproducts of transcription and are typically rapidly degraded after synthesis (Clark et al, 2012). (Albrecht et al, 2010(Albrecht et al, , 2011Chabelskaya et al, 2014;Chao et al, 2012;Gong et al, 2011;Grieshaber et al, 2006;Mann et al, 2012;Moon et al, 2013;Ortega et al, 2012;PadalonBrauch et al, 2008;Pfeiffer et al, 2007) crRNA, (Bhaya et al, 2011;Heidrich and Vogel, 2013;Sampson et al, 2013;van der Oost et al, 2014) (Georg and Hess, 2011;Giangrossi et al, 2010;GonzaloAsensio et al, 2013;Gottesman and Storz, 2011;Lasa et al, 2011;Lee and Groisman, 2010;Padalon-Brauch et al, 2008;Sesto et al, 2013) circRNAs rare varies arise from the 3'-5' ligation of both ends of linear RNA molecules n. a. -- (Doose et al, 2013;Vicens and Cech, 2009) - (Conway et al, 2014;Weinberg et al, 2009) Carpenter et al, 2013;Gomez et al, 2013;Iiott et al, 2014;Imamura et al, 2014;Li et al, 2014;Rapicavoli et al, 2013) poly ( …”

Section: Eukaryotic Long Non-coding Rnas (Lncrnas)mentioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…The dRNA-seq approach has already been validated in Helicobacter, Synechocystis, and other organisms (16,(28)(29)(30)(31), but not in S. Typhimurium. It was important to put our global TSS approach into the context of the wealth of the Salmonella literature.…”

Section: Identification Of Transcriptional Start Sites Under Infectiomentioning

confidence: 99%

The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium

Kröger¹,

Dillon²,

Cameron³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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380

427

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More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNAsequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ 70 (including phoP, slyA, and invF) from which we identified the −10 and −35 motifs of σ 70 -dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model. transcriptional mapping | noncoding RNA | posttranscriptional regulation | pathogenicity | genome sequence

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The transcriptional landscape of Chlamydia pneumoniae

Cited by 73 publications

References 58 publications

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Dual RNA-seq of pathogen and host

The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium

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